Non-obligate predatory bacterium burkholderia casidae and uses thereof

ABSTRACT

A novel predator bacterium  Burkholderia casidae  is disclosed. The invention is directed to the isolation and use of  Burkholderia casidae  to control microbial diseases of plants. The genetic, biochemical and physiological characteristics of  Burkholderia casidae  are described. Biocontrol compositions comprising  Burkholderia casidae , and antimicrobial compounds and antimicrobial preparations prepared from  Burkholderia casidae  are also disclosed, as are methods for accomplishing all of the foregoing.

[0001] This application is a continuation-in-part of provisionalapplication serial No. 60/044,532, filed Apr. 23, 1997, the disclosureof which is incorporated herein by reference in its entirety.

1. FIELD OF THE INVENTION

[0002] The present invention relates to predator bacteria that havebiocontrol activity against microorganisms. More particularly, thepresent invention is directed to a novel, non-obligate predatorbacterium Burkholderia casidae (including variants thereof). Theinvention is also directed to methods for isolating and producingsubstantially pure cultures of Burkholderia casidae, and toantimicrobial preparations produced from such cultures. The invention isfurther directed to biocontrol compositions comprising such purecultures, extracts and filtrates of such cultures, cell fractionsprepared from such cultures, and antimicrobial preparations producedfrom Burkholderia casidae. The invention is additionally directed tomethods for protecting plants against microbial diseases by treatmentwith biocontrol compositions of the invention.

2. BACKGROUND OF THE INVENTION

[0003] Past attempts to control plant microbial diseases have includedthe use of chemicals. However, many chemicals that have been inlong-time use are now ineffective due to the increasing number ofchemical-resistant strains of plant pathogens. Further, many suchchemicals are recognized to be potentially hazardous to non-targetorganisms, particularly humans, and to the environment.

[0004] Biological control of plant pathogens is an alternative tochemical control. It has been recognized that crops grown in some soilsnaturally are resistant to certain fungal pathogens. Furthermore, soilsthat are conducive to the development of fungal diseases can be renderedsuppressive, or resistant, by the addition of small quantities of soilfrom a suppressive field. Scher et al., Phytopathology 70:421 (1980).Conversely, suppressive soils can be made conducive to fungal diseasesby autoclaving or chemical fumigation, indicating that the factorsresponsible for disease control are biological. Root colonizing bacteriahave been shown to be responsible for this phenomenon, which is known asbiological disease control. Baker et al., Biological control of plantpathogens, Freeman Press, San Francisco (1974).

[0005] In many cases, the most efficient strains of biological diseasecontrolling bacteria are fluorescent Pseudomonads. Weller et al.,Phytopathology, 73: 463-469 (1983). Many biological control Pseudomonasstrains produce metabolites, such as antibiotics and sideophores, thatinhibit the growth of fungal pathogens. Howell et al., Phytopathology69: 480-482 (1979); Howell et al., Phytopathology 70: 712-715 (1980).

[0006] An important factor in biological control is the ability of anorganism to compete in a given environment. Thus, it is desirable toobtain novel biocontrol agents which effectively control (e.g., retard,restraint, kill, lyse) the growth of plant pathogens, particularlyfungi, and which are able to aggressively compete with indigenousbacteria and other microflora that can exist on the surfaces or therhizosphere of the plant.

3. SUMMARY OF THE INVENTION

[0007] The present invention generally relates to predator bacteria.More particularly, the invention is directed to a novel, non-obligatepredator bacterium Burkholderia casidae, which has biocontrol activityagainst a broad range of microorganisms, particularly microbialpathogens. One aspect of the invention is directed to substantially purecultures of B. casidae (including variants), and to methods forisolating and producing such substantially pure cultures. In a preferredembodiment, the invention provides a substantially pure culture of B.casidae strain 2.2N (ATCC accession no. 55961).

[0008] Another aspect of the invention is directed to preparations ofantimicrobial compounds produced by B. casidae, and to methods forproducing such antimicrobial preparations. In a preferred embodiment,the invention provides antimicrobial preparations comprising antifungaland anti-yeast compounds produced by B. casidae strain 2.2N or variantsthereof, and methods for producing such preparations.

[0009] An additional aspect of the invention is directed to biocontrolcompositions comprising materials obtained or derived from B. casidae,cells or cultures, and to methods for producing such biocontrolcompositions. In one embodiment, the biocontrol compositions compriselive B. casidae cells or cultures. In another embodiment, the biocontrolcompositions comprise inactivated B. casidae cells or cultures that mayhave been processed in a variety of ways including cell breakage andspray drying. In an additional embodiment, the biocontrol compositionscomprise cell-free filtrates or cell fractions prepared from B. casidaecells or cultures. In a further embodiment, the biocontrol compositionscomprise preparations of antimicrobial compounds isolated from B.casidae, cells or cultures, or cell-free filtrates or cell fractionsprepared from such cultures or cells. In preferred embodiments, thebiocontrol compositions comprise cells (living or dead) or cultures(living or dead) of B. casidae strain 2.2N, or cell-free filtrates orcell fractions, or antimicrobial, particularly anti-fungal, preparationsmade from such cells or cultures.

[0010] A further aspect of the invention is directed to methods for theprevention and/or treatment of plant microbial diseases using biocontrolcompositions of the invention. In one embodiment, the methods forprevention and/or treatment comprise treating the plants themselves withan effective amount of a biocontrol composition. In another embodiment,the methods for prevention and/or treatment comprise treating plantseeds prior to planting with an effective amount of a biocontrolcomposition. In a further embodiment, the methods for prevention and/ortreatment comprise treating the soil in the immediate vicinity of theplant with an effective amount of a biocontrol composition. In preferredembodiments, the methods of the invention are directed to preventionand/or treatment of plant fungal diseases using biocontrol compositionsof the invention.

[0011]B. casidae (including variants thereof) of the invention exertsbiocontrol activity against a wide spectrum of microorganisms. B.casidae adversely affects the growth or survival of such microorganismsin its vicinity. Because some of the antimicrobial activity of B.casidae is present in cell-free culture-filtrates, biocontrol activityis soluble in water and can exert its activity against microorganisms inaqueous suspension. Antimicrobial activity does not require living cellsor cell contact. Susceptible microorganisms are referred to herein asprey or targets of B. casidae. Prey of B. casidae include, but are notlimited to, fungi (including yeast), algae, protozoa, and bacteria(including blue green algae) other than B. casidae.

[0012]B. casidae exerts biocontrol activity against a particular preymicroorganism in its vicinity by releasing one or more antimicrobialcompounds that adversely affect the growth or survival of the prey,and/or by physically attacking the prey through attachment, subsequentparasitism and eventual destruction of the prey. Different preymicroorganisms are susceptible to different combinations of such actionsby B. casidae. Some prey are susceptible only to physical attack by B.casidae. Other prey are susceptible only to a particular one, orcombination, of the antimicrobial compounds released by B. casidae. Yetother prey are susceptible to both physical attack and one or more ofthe released antimicrobial compounds.

[0013] Since many prey microorganisms of B. casidae are pathogens, theantimicrobial preparations and biocontrol compositions of the inventionbeneficially may be used in the prevention and/or treatment of microbialdiseases of plants and animals including humans. In particular, theantimicrobial preparations of the invention may be used to manufacturemedicaments for use in treatment of microbial diseases of animals, fish,and humans, and to produce bactericides and fungicides for use incontrolling microbial diseases, particularly those of plants. Thebiocontrol compositions of the invention may be directly used to preventand/or treat microbial, particularly fungal, diseases of plants. Theantimicrobial compounds and biocontrol compositions of the inventionalso may be used to treat, or prevent, protozoan diseases, and tocontrol protozoan and algal growth in aquatic environments.

[0014] The present invention may be understood more fully by referenceto the following detailed description of the invention, examples ofspecific embodiments of the invention and the appended figure.

[0015] 3.1. Definitions

[0016] In order to provide a clear and consistent understanding of thespecification and claims, including the scope to be given to a term, thefollowing definitions are given to various terms and abbreviations usedherein.

[0017] 2.2N

[0018]Burkholderia casidae strain 2.2N (ATCC accession no. 55961); alsoreferred to as “strain 2.2N”.

[0019] antifungal

[0020] as used herein, the term “antifungal” includes anti-yeastactivity, unless indicated otherwise.

[0021] antimicrobial compound

[0022] A compound that has one or more adverse effects against amicroorganism, such as retarding, suppressing or stopping growth of themicroorganism or killing or lysing the microorganism. The adverse effecton growth may be temporary or permanent. An antimicrobial compound maybe more particularly an anti-filamentous fungi compound,anti-mycobacteria compound, anti-yeast compound, anti-algal compound,anti-protozoan compound and/or antibacterial compound.

[0023]B. casidae

[0024]Burkholderia casidae . As used herein, the term “B. casidae”,unless otherwise modified, encompasses the B. casidae species and all B.casidae strains and variants thereof.

[0025]B. casidae variant

[0026] A B. casidae variant is a mutant or derivative of a B. casidaestrain. The mutation in a mutant may be chromosomal or extrachromosomal,and may be spontaneous or artificially-induced. Derivatives include B.casidae strains and mutants that contain deletion or insertion of DNA.Also included are B. casidae strains and mutants that have lost abacteriophage (i.e., as prophage) or plasmid, or one or more segments ofa chromosomal, plasmid, or bacteriophage (i.e., as prophage) DNA thatmay be found in B. casidae. Derivatives can also include B. casidaestrains and mutants that contain one or more extrachromosomal geneticelements, such as an insertion sequence or transposon or a plasmid orbacteriophage.

[0027] BHIM

[0028] Brain Heart Infusion Medium, one-tenth strength Brain HeartInfusion Broth (Difco, Detroit, Mich.).

[0029] BHIM agar

[0030] BHIM with 1.5% agar.

[0031] biocontrol

[0032] The ability to retard or suppress the growth of a microorganism,or to kill or lyse the microorganism.

[0033] cell fractions

[0034] Cells can be broken to obtain, for example, the following cellfractions: cell walls, cell membranes, soluble material, and particulatematerial.

[0035] cell-free filtrate

[0036] Medium of culture from which cells have been removed by, forexample, filtration, centrifugation or precipitation.

[0037] cells

[0038] Cells recovered from culture.

[0039] culture

[0040] Cells with medium in which cells have been grown.

[0041] culture fractions

[0042] Cells and cell-free culture medium recovered from cultures.

[0043] cysts

[0044] Resting stage of cells with a thicker cell wall.

[0045] filter-sterile

[0046] A term used to describe a process applied to a solution, culturemedium, etc. that removes any microorganisms therefrom by passing suchliquid materials through a porous membrane or matrix and hence separatesthe cells from the liquid.

[0047] fungi

[0048] The term includes filamentous fungi and yeast.

[0049] GSM

[0050] Glutamate Synthetic Medium, 0.1% L-glutamic acid, 0.05%MgCl₂.6H₂O, 0.1% KH₂PO₄, 0.1% NH₄NO₃, 0.02% Na₂SO₄, and 0.02% NaCl (pH7.0).

[0051] HIB

[0052] Heart Infusion Broth (Difco, Detroit, Mich.).

[0053] microorganism

[0054] A bacterium, fungus (including yeast), alga, or protozoan whosecells can be best viewed by magnification (i.e., a microscope).

[0055] nitrogen-limiting

[0056] The condition where the nitrogen nutrient of the medium is, orwould be, exhausted before the exhaustion of any other essentialnutrients, such as carbon.

[0057] pasteurized

[0058] Process of heating (i.e., 80° C. for approximately 15 min) tokill cells; process can be applied to any culture or cell fraction.

[0059] PCR

[0060] Polymerase chain reaction.

[0061] prey

[0062] A prey microorganism. A microorganism whose growth or survival isadversely affected by a predator bacterium, such as B. casidae, presentin its immediate vicinity. The adverse effect may be due to physicalattack (contact) or release of one or more antimicrobial compounds bythe predator bacterium. Also known as target organism.

[0063] rRNA

[0064] Ribosomal RNA.

[0065] soil eluate

[0066] The water or media wash of a soil sample. Also referred to as“percolation” fluid.

[0067] spp.

[0068] Plural of species.

[0069] substantially pure culture

[0070] A culture of subject microorganism that is substantially free ofany contaminant microorganisms (i.e., other types or species ofmicroorganisms). Specifically, a substantially pure culture is at least75% (i.e., containing less than one contaminant microorganism per threesubject microorganism), preferably at least 90%, more preferably atleast 99.9%, and most preferably 100%, free of any contaminantmicroorganisms.

[0071] TSM

[0072] One-quarter strength Tryptic Soy Broth (Difco, Detroit, Mich.).

[0073] TSM+S

[0074] TSM with 0.2% (w/v) sucrose.

[0075] yeast

[0076] The term does not include filamentous fungi.

4. BRIEF DESCRIPTION OF THE FIGURE

[0077]FIG. 1 depicts the partial nucleotide sequence (from positions 27to 1522; approximately 98%) of 16S rRNA (SEQ ID NO: 1) of Burkholderiacasidae strain 2.2N, which was PCR amplified from genomic DNA usingprimers 27f (SEQ ID NO: 2) and 1522r (SEQ ID NO: 3).

5. DETAILED DESCRIPTION OF THE INVENTION

[0078] The present invention relates to a novel, non-obligate predatorbacterium, Burkholderia casidae, that has biocontrol activity against awide spectrum of microorganisms. The invention provides substantiallypure cultures of B. casidae (including variants thereof), as well asmethods for producing and recovering such cultures. The invention alsoprovides purified antimicrobial compounds produced by B. casidae. Theinvention further provides biocontrol compositions comprising B. casidaecells or cultures, cell-free filtrates or cell fractions of such cellsor cultures; or antimicrobial preparations made from such culturescells, or cell fractions; and methods for producing said biocontrolcompositions. The invention further provides methods for preventing ortreating plant microbial diseases by the application of biocontrolcompositions of the invention.

[0079] A specific embodiment of the invention, B. casidae strain 2.2N,has at least four distinct antimicrobial activities: (1) antibacterial,(2) anti-mycobacterial, (3) anti-filamentous fungi, and (4) anti-yeast.The distinction between the anti-filamentous fungi activity and theanti-yeast activity is based on differences in the heat-stability ofeach activity in cell-free materials, such as filtrates, obtained fromstrain 2.2N cultures (i.e., the anti-yeast activity is relatively moreheat-resistant). In addition, strain 2.2N produces one or more proteasesthat have broad spectrum antimicrobial activities. Strain 2.2N also hasanti-algal and anti-protozoan activities.

[0080] 5.1. Burkholderia casidae Strains

[0081]B. casidae is a naturally occurring, gram-negative soil bacterium.Its existence and isolation had not been previously described. Whencultivated in artificial media, B. casidae can exist in at least twoalternate structural forms. B. casidae exists in the so called cell formwhen it is grown in artificial media that are not limited for nitrogennutrients. The cell form of B. casidae is a short, motile rodapproximately 1 μm in length and approximately 0.5 μm in diameter. B.casidae also can exist in the so-called cyst form when cultivated inartificial medium. The cyst form of B. casidae is a spherical structureapproximately 1.25 μm in diameter and comprises a central bodyapproximately 0.25 μm in diameter and a coat that surrounds the centralbody. B. casidae cells differentiate into cysts when nitrogen nutrientsin the media become growth limiting. The cyst form of B. casidae is notmore heat or desiccation resistant than the cell form. Heat or chemicaltreatment of B. casidae cysts significantly increases the release rateof antimicrobial, particularly anti-fungal, compounds from the cysts.

[0082]B. casidae exhibits biocontrol activity against a wide spectrum ofprey microorganisms. As discussed above, the biocontrol activity of B.casidae against a particular prey microorganism may be due to physicalattack on the prey and/or the release of one of more antimicrobialcompounds that adversely affect the prey. B. casidae cell produces arange of antimicrobial compounds that are (1) stored intracellularly,(2) associated with cells, cysts, or cell and cyst fractions (e.g., cellwalls), or (3) are found in the medium outside of, and not associatedwith, cells due to either active export, secretion, or cellular lysis.

[0083] Fungi that are prey of B. casidae include saprophytes as well aspathogens of plants, animals and humans. Prey fungi include, but are notlimited to, those in any of the following genera: Agaricus, Alternaria,Aspergillus, Botrytis, Candida, Cercospora, Cercosporidium,Cryptococcus, Geotrichum, Mycosphaerella, Mucor, Penicillium, Phoma,Phytophthora, Plasmopora, Pseudopeziza, Puccinia, Pythium, Rhizoctonia,Rhizopus, Saccharomyces, Septoria, Sporothrix, Stemphylium,Trichophyton, and Verticillium. Bacterial prey of B. casidae include,but are not limited to, those in any of the following genera: Agromyces,Arthrobacter, Micrococcus, Mycobacterium, Nocardia, Staphylococcus, andStreptomyces. Algal prey of B. casidae can be eukaryotic or prokaryoticand include, but are not limited to, those of the Anabena spp. Protozoanprey of B. casidae include, but are not limited to, those of theParamecium and Tetrahymena spp.

[0084] A preferred embodiment of the invention is B. casidae strain2.2N. In addition to having the above described biological andbiocontrol characteristics, strain 2.2N also has the followingparticular genetic, biochemical, cultural, enzymatic, and metaboliccharacteristics.

[0085] Strain 2.2N has a 16S rRNA gene sequence comprising that of SEQID NO: 1. As displayed in Table 3, infra, pairwise comparisons of 16SrRNA gene sequences show that strain 2.2N shares high sequencesimilarity with members of the genus Burkholderia (i.e., >85%). Bycontrast, Escherichia coli gave a similarity value of 71.7% (Table 3).The 16S rRNA data indicate strain 2.2N belongs to the Burkholderiagenus.

[0086] Organisms sharing more than 97% rRNA similarity belong to asingle species (Vandamme et al., 1996, Microbiol. Reviews 60:407-438).The 16S rRNA of strain 2.2N shares less than 95% sequence similaritywith the 16S rRNA of known Burkholderia spp. Thus, strain 2.2Nrepresents a novel Burkholderia species.

[0087]B. casidae strain 2.2N has a cellular fatty acid composition thatdiffers markedly from those of other of Burkholderia species.Specifically, strain 2.2N has significantly less C16:0 fatty acids,almost no 16:1 fatty acids, almost no C17:CPA (cyclopropanylated) fattyacids, and high levels of both C18:1 (9,10 and 11,12) fatty acids and2-OH C15:0 fatty acids (see Table 4, infra). This pattern appears to beunique among known Burkholderia spp., further supporting the conclusionthat strain 2.2N represents a novel Burkholderia spp.

[0088] As assayed with the API50 CH test (bioMerieux Vitele, Inc.,Hazelwood, Mo.), strain 2.2N can utilize all of the following compoundsas substrates: adipate, caprate, D-fucose, galactose, glucose, mannitol,mannose, phenylacetate and sucrose as substrate; and cannot utilize anyof the following compounds as substrate: 2-ketogluconate, cellobiose,D-arabinose, D-xylose, glycerol, inositol, L-arabinose, malate,rhamnose, sorbitol, and trehalose.

[0089] As assayed with the API-NFT test (bioMerieux S. A.,Marcy-l'Etiole, France), strain 2.2N can grow at about 41° C., reducenitrate to nitrite, hydrolyze esculin and gelatin, and form indole.Strain 2.2N cannot produce yellow pigments, urease, arginine dihydrolaseand cytochrome oxidase.

[0090] As assayed with the Sceptor Pseudomonas/Resistant MIC Panel(Becton-Dickinson, Sparks, Nev.), strain 2.2N is resistant to theantibiotics amikacin, cefoperazone, gentamycin, tetracycline,ticarcillin/clavulanic acid and tobramycin; and is susceptible to theantibiotics cefotaxime, ceftizoxime, ceftazidime, ceftriaxone,ciprofloxacin, imipenem, and trimethoprim/sulfamethoxazole.

[0091] A pure culture of B. casidae strain 2.2N was deposited on Apr.23, 1997 with the American Type Culture Collection (ATCC), now locatedat 10801 University Boulevard, Manassas, Va. 20110-2209, in compliancewith the provisions of the Budapest Treaty on the InternationalRecognition of the Deposit of Microorganisms for the Purpose of PatentProcedure, and has been assigned ATCC accession number 55961.

[0092] The present invention contemplates all B. casidae strains andvariants thereof. According to the invention, a B casidae strain hasbiocontrol, genetic, biochemical and metabolic characteristics that aresubstantially similar to those of strain 2.2N. Specifically, a B.casidae strain has a 16S rRNA gene comprising a sequence that is atleast 97%, preferably at least 99%, and most preferably 100%, identicalto the 16S rRNA gene sequence of strain 2.2N, which comprises thesequence of SEQ ID NO: 1, as determined by Clustal Analysis.

[0093] A B. casidae strain also has cellular fatty acid content that issubstantially similar to that of strain 2.2N, as shown in Table 4,infra. Specifically, a B. casidae strain has a fatty acid compositionwherein (a) C16:0 fatty acid comprises from about 16% to about 20%,preferably about 18%, of the total cellular fatty acid content; (b)C16:1 fatty acid comprises from about 18% to about 22%, preferably about21%, of the total cellular fatty acid content; and (c) C18:1 (11,12)fatty acid comprises from about 35% to about 45%, preferably about 39%,of the total cellular fatty acid content.

[0094] As assayed with the API50 CH test (bioMerieux S. A.,Marcy-l'Etiole, France), a B. casidae strain can utilize at least seven,preferably at least eight, and most preferably nine, of the followingcompounds as substrate: adipate, caprate, D-fucose, galactose, glucose,mannitol, mannose, phenylacetate and sucrose; and can utilize no morethan two, preferably no more than one, and most preferably none, of thefollowing compounds as substrate: 2-ketogluconate, cellobiose,D-arabinose, D-xylose, glycerol, inositol, L-arabinose, malate,rhamnose, sorbitol, and trehalose.

[0095] 5.1.1. Antimicrobial Compounds Produced by Burkholderia casidae

[0096]B. casidae produces and releases one or more antimicrobialcompounds. Such compounds may achieve their adverse effects on a preymicroorganism by acting alone or in combination with other antimicrobialcompounds produced by B. casidae.

[0097]B. casidae produces one or more antimicrobial compounds that eachalone or in combination have predominately anti-bacterial activity. Suchantibacterial compounds are active against bacteria that include, butare not limited to, those from any of the Agromyces, Arthrobacter,Micrococcus, Mycobacterium, Nocardia, Staphylococcus, and Streptomycesgenera. The antibacterial compounds produced by B. casidae arerelatively heat-labile. For example, incubation of crude preparations ofsuch compounds at about 80° C. for approximately 10 minutes causesapproximately 100% loss of antibacterial activity.

[0098]B. casidae also produces one or more antimicrobial compounds thateach alone or in combination have predominately anti-filamentous fungiactivity. Such anti-filamentous fungi compounds collectively are activeagainst filamentous fungi that include, but are not limited to, thosefrom any of the following genera: Agaricus, Alternaria, Aspergillus,Botrytis, Cercospora, Cercosporidium, Cryptococcus, Geotrichum, Mucor,Penicillium, Phoma, Phytophthora, Plasmopora, Pseudopeziza, Puccinia,Pythium, Rhizoctonia, Rhizopus, Septoria, Sporothrix, Stemphylium,Trichophyton, and Verticillium. The anti-filamentous fungi compoundscollectively also are active against Mycobacterium spp. and Anabena spp.In comparison to the antibacterial compounds produced by B. casidaecells, the anti-filamentous fungi compounds are relatively heat-stable.For example, crude preparations of anti-filamentous fungi compoundsproduced by B. casidae can withstand incubation at approximately 80° C.for about 10 minutes and suffer less than 35% loss of anti-filamentousfungi activity.

[0099]B. casidae also produces one or more antimicrobial compounds thateach alone or in combination have predominantly anti-yeast activity.Such anti-yeast compounds are active against yeast that include, but arenot limited to, those from any of the following genera: Candida,Cryptococcus, and Saccharomyces. In comparison to the antibacterial andthe anti-filamentous fungi compounds produced by B. casidae, theanti-yeast compounds produced are very heat-resistant. For example,crude preparations of anti-yeast compounds produced by B. casidae canwithstand incubation at approximately 80° C. to 100° C. for about 10minutes and incur less than 5% loss of anti-yeast activity.

[0100]B. casidae cysts produce and accumulate anti-filamentous fungi andanti-yeast compounds. The cysts release such compounds at a relativelyslow rate. The release rate can be increased significantly by inducingthe cysts to grow, particularly by incubating the cysts in fresh,nitrogen-rich media at about 28° C. The rate of release also can beincreased by exposing the cysts to boiling or autoclaving, and/or bytreating the cysts with various organic solvents, such as alcohols.

[0101] Except under special conditions (see below), B. casidae culturesmay comprise mixtures of cells and cysts. Further, the relative amountsof cells and cysts in such cultures may change in response to changingculture conditions (e.g., the relative levels of carbon and nitrogennutrients). Accordingly, most B. casidae cultures, through theirlife-cycle, typically produce all the antibacterial, anti-yeast andanti-filamentous fungi compounds that are associated with cells or foundin the medium.

[0102] 5.2. Cultures of Burkholderia casidae and Variants

[0103] 5.2.1. Isolation and Purification of B. casidae

[0104] The present invention provides substantially pure cultures of B.casidae (including variants thereof). Such substantially pure culturesmay be obtained by a method comprising: isolating non-obligate predatorbacteria, which include B. casidae, from soil; purifying the predatorbacteria isolates; screening and identifying the isolates for those thathave the unique genetic and biochemical characteristics of B. casidae;and growing the B. casidae isolates, thereby producing substantiallypure cultures of B. casidae.

[0105] The isolation, identification, and production of substantiallypure cultures of B. casidae are carried out using standardmicrobiological techniques. Examples of such techniques may be found inGerhardt, P. (ed.) Methods for General and Molecular Microbiology.American Society for Microbiology, Washington, D. C. (1994) andLennette, E. H. (ed.) Manual of Clinical Microbiology, Third Edition.American Society for Microbiology, Washington, D. C. (1980).

[0106] Non-obligate, predator bacteria can be isolated from manydifferent types of soil. Preferably the soil is from the temperate orsubarctic region in North America. Useful soils include various types ofloam soil, such as silty clay loam, silt loam, clay loam and loam. Apreferred source for isolating of B. casidae is loam type soil fromfallow agricultural fields.

[0107] The isolation of B. casidae from soil optionally can be precededby treatment of the source material with one or more mutually,non-exclusive enrichment procedures that increase the absolute number orrelative amount of B. casidae in the sample material. The sourcematerial may be subjected to a single enrichment procedure or severaldifferent enrichment procedures in sequence, prior to use in isolatingB. casidae.

[0108] In one enrichment procedure, a soil sample or soil eluate (i.e.,an aliquot of a water or media wash of the soil sample) is incubatedwith an inoculum of a prey microorganism of B. casidae for several days,at about 28° C., under conditions conducive to the growth of B. casidae(see below for such conditions). The prey microorganism serves as a foodsource for B. casidae and thereby increases the level of B. casidae inthe source material. In one embodiment, the prey microorganism used inthe enrichment procedure is Micrococcus luteus. In another embodiment,the prey microorganism is Saccharomyces cerevisiae.

[0109] In another enrichment procedure, a soil sample or soil eluate isplated on a copper-containing agar medium and incubated at about 28° C.for several days to select for copper-resistant bacteria, which includeB. casidae. Colonies that grow on a copper-containing medium arecandidates for predator testing and/or screening for B. casidae. In oneembodiment of this procedure, the selection agar medium contains 0.25%(w/v) Brain Heart Infusion Broth (Difco), 0.01% (w/v) CuCl₂.2H₂O and1.5% (w/v) agar pH 6.5.

[0110] In yet another enrichment procedure, a soil sample or soil eluateis plated on Noble agar containing a small amount (e.g., about 1% [v/v])of Arthrobacter globiformis culture filtrate and incubated at 28° C. forseveral days to select for bacteria that can grow on such a medium.Colonies that have a “fried-egg” or “beehive” appearance on this mediumare candidates for predator testing and/or screening for B. casidae.

[0111] In yet a different enrichment procedure, a soil sample or soileluate is plated on minimal medium that lacks a carbon source andcontains only a minimal level of nitrogen nutrients and incubated forseveral days to select for bacteria that can grow on such medium.Colonies that have a “fried-egg” or “beehive” appearance are preferredcandidates for subsequent predator testing and/or screening for B.casidae. In an embodiment, the enrichment procedure uses a modified BurkAzotobacter medium comprising 0.08% K₂HPO₄, 0.02% KH₂PO₄, 0.02%MgSO₄.7H₂O, 0.01% CaSO₄.2H₂₀O 0.25 mg/L NaMoO₄.2H₂O, 8.6 mg/LFe(NH₄)₂(SO₄)₂.12H₂O, and 1.5% Noble agar.

[0112]B. casidae may be isolated by first isolating predator bacteriafrom a source material. The source material may be a untreated soil orsoil eluate, or a enriched product of such sample materials prepared asdescribed above. Predator bacteria can be isolated by plating a smallamount of the source material on an agar medium previously inoculatedwith a sparse lawn of a prey microorganism of B. casidae, and examiningfor colonies that are surrounded by a zone of clearing or inhibition ofthe prey microorganism after several days of incubation at about 28° C.The sparse lawn of prey microorganism may be prepared in the form of atop-agar overlay or a spread of a bacterial inoculum on a base agarmedium that can support growth of the prey. In one embodiment, the preymicroorganism is M. luteus and the base agar medium is BHIB agar (0.25%(w/v) Brain Heart Infusion Broth, 1.5% (w/v) agar). In anotherembodiment, the prey microorganism is Staphylococcus aureus and the baseagar medium is BHIM agar. In yet another embodiment, the prey organismis S. cerevisiae and the base agar medium is BHIM agar.

[0113] Bacterial colonies that produce a zone of clearing or inhibitionof prey microorganism are tested for predator activity by streaking asample of the colony perpendicularly across a streak of a preymicroorganism on a plate of agar medium that can support the growth ofthe prey, and incubating the test plate at about 28° C. for severaldays. Predator bacteria are those whose growth spreads and tracks alongwith the track of the prey microorganism and lyses the preymicroorganism at the intersections of the bacterial streaks. In oneembodiment, the predator test uses M. luteus as the prey microorganism.In another embodiment, the test uses S. cerevisiae as the prey organism.

[0114] A predator bacteria isolate may be purified from any contaminantmicroorganism using any technique known in the art. For example, apredator bacteria isolate may be purified by several rounds, preferablyat least three rounds, of streaking or plating the isolate from asingle, isolated colony. Agar media that may be used for the isolation,purification and maintenance of B. casidae strains include, but are notlimited to, HIB agar, BHIM agar, TSM agar (TSM with 1.5% agar) and theirequivalents.

[0115] To isolate B. casidae, the purified predator bacteria isolatesare screened for those that have the biological, genetic, biochemicaland/or enzymatic characteristics of B. casidae. According to theinvention, a predator bacteria is B. casidae if it has the 16S rRNA genesequence and the cellular fatty acid profile of B. casidae as describedin Section 5.1, above.

[0116] 5.2.1.1. B. casidae Variants

[0117] The invention also provides variants of B. casidae, whichcomprise naturally-occurring and artificially-constructed mutants andderivatives of B. casidae . The B. casidae variants have substantiallythe same 16S rRNA gene sequence (i.e., ≧95% identity) and cellular fattyacid profile as those described in Section 5.1, above, for B. casidae.Variants may have a different number of rRNA cistrons as well. B.casidae variants of the invention may vary in their biocontrol activityand/or production of antimicrobial compounds as compared to, forexample, wild-type strain 2.2N. Variants also may have differentcolonial morphology, colonial pigmentation, and growth ratecharacteristics. Variants may also have a different spectrum ofantimicrobial activities and enzymatic activities. Preferred B. casidaevariants of the invention are those that have biocontrol activityidentical or superior to that of the corresponding wild-type B. casidaestrain (e.g., strain 2.2N). As used herein, superior biocontrol activitymeans an adverse effect on a wide spectrum of prey microorganisms,and/or production of a higher level and/or greater number ofantimicrobial compounds.

[0118] Naturally-occurring B. casidae variants may be isolated fromsoil, purified and identified using approaches and methods described insection 5.2.1. above. Spontaneous B. casidae variants may be isolated byscreening a B. casidae strain on agar media for variants in colonymorphology and/or inhibitory activity against a prey microorganism. Inan embodiment, B. casidae variants may be isolated by screening strain2.2N.

[0119]B. casidae variants also may be artificially constructed from B.casidae strains (e.g., strain 2.2N) using methods known in the art ofbacterial genetics and recombinant DNA technology. For example, B.casidae mutants may be produced by mutagenizing B. casidae strains withchemical mutagens or irradiation, and selecting or screening forisolates that have the desired alterations in phenotype and/or genotype(e.g., biocontrol activity against one or more prey microorganism). B.casidae derivatives may be produced from B. casidae strains by bacterialconjugation; transformation with DNA, including plasmid and phage DNA;or transduction or transfection with phages. B. casidae derivatives mayalso be produced as a consequence of the introduction of insertionsequences (IS) or transposons (Tn) via conjugation, transformation, ortransduction. The following references provide exemplary teachings ofgenetic and/or recombinant DNA techniques that may be used to constructsuch B. casidae variants: Gerhardt, P. (ed.) Methods for General andMolecular Microbiology. American Society for Microbiology, Washington,D. C. (1994); Berger and Kimmel, Guide to Molecular Cloning Techniques,Methods in Enzymology, Volume 152. Academic Press, NY, (1987); Cheng andLessie, 1994, “Multiple replicons constituting the genome of Pseudomonascepacia,” J. Bacteriol. 176:4034-4042; Simon et al., “A broad host rangemobilization system for in vivo genetic engineering: transposonmutagenesis in Gram-negative bacteria,” Bio/Technology, November 1983,pages 784-791; Darzins and Chakrabarty, 1984, “Cloning of genescontrolling alginate biosynthesis from a mucoid cystic fibrosis isolateof Pseudomonas aeruginosa,” J. Bacteriol. 159:9-18; Irani and Rowe,1997, “Enhancement of transformation in Pseudomonas aeruginosa PAO1 byMg²⁺ and heat,” BioTechniques 22:45-56; Lennon and DeCicco, 1991,“Plasmids of Pseudomonas cepacia strains of diverse origin,” AppliedEnviron. Microbiol. 57:2345-2350; and U.S. Pat. No. 5,639,949.

[0120] 5.2.2. Growth of B. casidae

[0121]B. casidae may be grown aerobically at about 20° C. to about 41°C., preferably at about 28° C. to about 30° C., on any solid or liquidmedium that will support its growth. Useful media for growing B. casidaeinclude, but are not limited to, BHIM, TSM+S, and MSB medium composed of1% molasses (or 1% corn syrup) and 1% soy bean meal (or 1% peanut mealor 1% cotton seed meal). MSB medium and equivalents are particularlyuseful for production of large volume cultures because of their lowcost. Synthetic media such as GSM may also be used to grow B. casidae.

[0122] The invention provides cultures of B. casidae cells (in contrastto B. casidae cysts). According to the invention, cultures of B. casidaecells contain at least 80%, preferably at least 95%, and most preferablyat least 99%, cells. Cultures of B. casidae cells can be prepared bygrowing B. casidae in media that are not nitrogen-limiting, andharvesting the cultures at any time from early log phase to stationaryphase. Media that have balanced carbon and nitrogen nutrients are notnitrogen-limiting. Such media are especially useful for producingcultures of B. casidae cells. Examples of balanced carbon and nitrogennutrient media include GSM and media composed essentially of preymicroorganisms of B. casidae.

[0123] Cultures of B. casidae cells also can be prepared by growing B.casidae in any medium that will support their growth, and harvesting thecultures before nitrogen nutrients become limiting. Media that can beused in this manner to produce cultures of cells include BHIM, TSM andMSB. The optimal time for harvesting cultures of B. casidae cells grownin such media can be determined by monitoring the culture's cell andcyst content by microscopy. Typically, log phase is the optimal time forharvesting cultures of B. casidae cells grown in such media.

[0124] The invention also provides cultures of B. casidae cysts.According to the invention, cultures of B. casidae cysts contain atleast 80%, preferably at least 95%, and most preferably at least 99%percent cysts.

[0125] Cultures of B. casidae cysts can be prepared by growing B.casidae in media that are nitrogen-limited and harvesting the culturesafter nitrogen nutrients become limiting or have been exhausted. Mediathat can be used for producing cultures of cysts include TSM+S. Theoptimal time for harvesting cultures of B. casidae cysts grown in suchmedia can be determined by monitoring cultures' cell and cyst content.Typically, stationary phase is the optimal time for harvesting culturesof cysts produced in such media.

[0126] Cultures of B. casidae cysts also can be prepared by growing orincubating at 28° C., B. casidae cells in media that arenitrogen-deficient and harvesting the cultures after any residual mediaand intracellular nitrogen nutrients have been exhausted. Media that canbe used for producing cultures of cysts in this manner include Burk'sBroth (0.064% [w/v] K₂HPO₄, 0.016% [w/v] KH₂PO₄, 0.005% [w/v]CaSo₄.2H₂O, 0.02% [w/v] MgSO₄.7H₂O, 0.02% [w/v] NaCl, 1.0% [w/v]sucrose). The optimal time for harvesting cultures of cysts from suchmedia can be determined by monitoring the cultures for their cystcontent by microscopy. Typically, the optimal time for harvestingcultures of cysts produced in such media is after growth has ceased andthe cultures are in the stationary phase of growth.

[0127] Cultures of B. casidae grown and harvested under conditions otherthan those described above may contain vegetative cells and/or cysts.The vegetative cell and cyst contents of the cultures can be determinedby microscopy.

[0128] 5.3. Antimicrobial Preparations

[0129] The invention provides antimicrobial preparations containingantifungal (including anti-filamentous fungi and anti-yeast) compoundsproduced by B. casidae. The antimicrobial preparations of the inventioncomprise alcohol-extracts of B. casidae cells, cysts, culture,suspension, cell-free filtrate or cell fraction, which alcohol-extractscan be prepared by extracting any of the foregoing materials with analcohol and concentrating the alcohol soluble material.

[0130] In an embodiment, the antimicrobial preparation can be producedby a method comprising boiling an alcoholic mixture comprising the cell,culture, suspension, cell-free filtrate or cell fraction and an alcohol;clarifying the boiled mixture; mixing the boiled mixture with magnesiumsilicate (talc); collecting the magnesium silicate; washing themagnesium silicate with water; and eluting antifungal compounds from themagnesium silicate with an alcoholic solution. One skilled in the artcan determine the optimal conditions for each of these steps, and indeedthe entire procedure, by varying each step in a systematic fashion andassaying for the antimicrobial activity recovered under each variationusing routine assay procedures.

[0131] In a preferred embodiment, a method for producing theantimicrobial preparation of the invention comprises the following:Isopropanol is added to a B. casidae culture, or its cell-free filtrateor cell fraction, or a suspension of B. casidae cells and/or cysts to afinal concentration of about 47% (v/v). The solution is boiled for ashort period and then cooled. The extraction solution is clarified bycentrifugation and concentrated three-fold by vacuum evaporation at 65°C. Magnesium silicate is added to the concentrated extraction solutionto approximately 8% (w/v) and the suspension stirred for about 30 min atroom temperature. The magnesium silicate is collected and washed withwater. Absorbed materials, including antifungal compounds, are elutedfrom the washed magnesium silicate with 70% isopropanol solution anddried by vacuum evaporation at 65° C. and thereby yielding theantimicrobial preparation.

[0132] The antimicrobial preparations of invention are soluble in wateror alcohol and have biocontrol activity against substantially the samespectrum of filamentous fungi and yeast as that described above for B.casidae culture.

[0133] 5.4. Biocontrol Compositions

[0134] The invention also provides biocontrol compositions that can beused to treat or prevent various microbial diseases of plants, animalsand humans, and to control the growth of A,algae and protozoa in aquaticenvironments. Biocontrol compositions of the invention comprise B.casidae cells, cysts or cultures; cell-free filtrates or cell fractionsof B. casidae cultures, cells or cysts; spray- or freeze-dried culturesor cell pastes (i.e., isolated cells); or antimicrobial preparationsproduced from B. casidae cultures, cells, cysts, cell-free filtrates orcell fractions.

[0135] The biocontrol compositions of the invention contain essentiallyone or several of three different types of active ingredients. One typeof active ingredient is the bacterium B. casidae itself, which can exertbiocontrol activity by physically attacking a prey microorganism and/orby the presence of extracellular antimicrobial compounds that areantagonistic to the prey microorganism. A second type of activeingredient is inactivated B. casidae cells or cysts, which exertbiocontrol activity through the release of intracellularly storedantimicrobial compounds. A third type of active ingredient is theantimicrobial compounds present in the medium of B. casidae culture orreleased from cells. A culture or suspension of live B. casidae containsthe first and third types of active ingredients. A culture or suspensionof inactivated B. casidae contains the second and third types of activeingredients. A cell-free filtrate or cell fraction of B. casidaeculture, or cell or cyst suspension, contains the third type of activeingredient.

[0136] 5.4.1. Liquid Biocontrol Compositions

[0137] The biocontrol compositions of the invention may be in form of aliquid or solid.

[0138] Liquid biocontrol compositions of the invention comprise liquidcultures of B. casidae; suspensions of B. casidae cells and/or cysts;cell-free filtrates or cell fractions of B. casidae cultures orsuspensions; suspensions of spray- or freeze-dried B. casidae culturesor cells; and/or antimicrobial preparations.

[0139] In one embodiment, liquid biocontrol compositions comprise liquidcultures or suspensions of live B. casidae. Preferably, such biocontrolcompositions comprise cultures or suspensions of B. casidae strain 2.2N.More preferably, such biocontrol compositions comprise stationarycultures of strain 2.2N grown in a nitrogen-limited medium, such asTSM+S, or suspensions of strain 2.2N harvested from such cultures.

[0140] In another embodiment, liquid biocontrol compositions compriseinactivated (i.e., sterilized) cultures or suspensions of inactivated B.casidae. According to the invention, cells of an inactiviated culture orsuspension cannot reproduce or divide. Preferably, the biocontrolcompositions of the invention comprise inactivated cultures orsuspensions of B. casidae strain 2.2N. More preferably, such biocontrolcompositions comprise inactivated stationary cultures of strain 2.2Ngrown in a nitrogen-limited medium, such as TSM+S, or inactivatedsuspensions of strain 2.2N harvested from such cultures. B. casidae maybe inactivated prior, or subsequent, to the harvest step.

[0141]B. casidae cells, cultures, suspensions, cell filtrates and cellfractions may be inactivated (i.e., treated such that any bacteriapresent no longer can divide or reproduce) using any method known in theart. Such method may comprise treating B. casidae cells, cultures,suspensions, cell filtrates and cell fractions with heat and/orchemicals to inactivate any live bacteria. Useful heat treatmentsinclude pasteurization, spray-drying at 150-200° C., and incubating aculture or suspension or cell-filtrate or cell fraction at about 80° C.to 100° C. for about 1 to about 15 minutes.

[0142] Cells, cultures, suspensions, cell filtrates and cell fractionsof B. casidae also may be treated with various types of alcohols toinactivate any live bacteria. Alcohols that may be used to inactivate B.casidae include methanol, ethanol and isopropanol. In specificembodiments, the aforementioned materials are treated with isopropanol,which is added to a final concentration of about 70% (v/v), and thealcohol solution is incubated for about 15 min to about 1 hour attemperatures ranging from about 40 to 25° C. At the completion of theinactivation process, alcohol may be optionally removed from thesolution or suspension by vacuum evaporation at 65° C.

[0143] Biocontrol compositions comprising B. casidae suspensions mayalso be prepared by harvesting B. casidae grown on solid media, andresuspending the harvested cells in buffers or fresh bacterial media.Such suspensions may be used directly in the preparation of biocontrolcompositions or after B. casidae has been inactivated. Such suspensionscan be treated using methods and conditions described above for the“inactivation” of liquid cultures of B. casidae.

[0144] The invention provides liquid biocontrol compositions comprisingB. casidae cultures or suspensions that have combined cell and cystconcentrations of about 1×10³ to about 3×10¹¹ cells and cysts per ml,preferably about 1×10⁶ to about 3×10¹¹ cells and cysts per ml, and mostpreferably about 1×10⁸ to about 3×10¹¹ cells and cysts per ml. Theconcentration of cells and cysts of B. casidae in liquid biocontrolcompositions may be adjusted using methods known in the art, e.g., bydiluting with buffers or bacterial media or by concentrating withfiltration or centrifugation.

[0145] In yet additional embodiments, liquid biocontrol compositions ofthe invention comprise cell-free filtrates or cell fractions of B.casidae cultures or suspensions having cell and/or cyst concentrationsdescribed above. Cell-free filtrates or cell fractions may be preparedfrom such cultures and suspensions of live or inactivated B. casidae(prepared as described above) by standard methods such as centrifugationfollowed by filtration of the supernatant. In preferred embodiments, thecell-free filtrates or cell fractions are prepared from inactivated B.casidae cultures or suspensions which have been incubated for severaldays in media or buffer to accumulate the antimicrobial compoundsextracellularly. The concentration of antimicrobial compounds present inthe cell-free filtrates or cell fractions may be adjusted by dilutingwith buffers or media or by concentrating with vacuum evaporation orultrafiltration.

[0146] In additional embodiments, liquid biocontrol compositions of theinvention comprise spray- or freeze-dried cultures, cells (e.g., cellpastes), cell-free culture-filtrates, or cell- or culture-fractions.Spray drying can be performed at temperatures of 150° to 200° C. withoutloss of biocontrol activity.

[0147] In further embodiments, the liquid biocontrol compositions of theinvention comprise solutions of antimicrobial preparations prepared asdescribed in section 5.3. above.

[0148] As an important use of the liquid biocontrol compositions of theinvention is for treatment of plants (discussed below) to protectagainst or treat microbial diseases, the biocontrol compositions maycomprise additional ingredients known in the art that facilitate suchtreatments or enhance the effectiveness of such treatments. Suchadditional ingredients include, but are not limited to, antioxidants,dyes, detergents, salts, emulsifiers, surfactants, encapsulants (e.g.,cellulose- and lignin-based gels), inert carriers (e.g., diatomaceousearth, vermiculite, and clay minerals), ultraviolet (UV) light-blockingagents, preservatives, and thickening agents (e.g., gelatin,polyethyleneglycol). Such additional ingredients should not be used atlevels that would be phytotoxic to the plant species being treated and,preferably, also not inhibitory to B. casidae where live cultures orcells or cysts are being applied in the treatment.

[0149] 5.4.2. Solid and Gel-Encapsulated Biocontrol Compositions

[0150] Solid biocontrol compositions of the invention comprise solidcarriers containing, or coated with, any of the following: live orinactivated B. casidae; antimicrobial compounds produced by B. casidae;and antimicrobial preparations of the invention. Numerous solid carriersare known in the art and may be used. They include, but are not limitedto: activated charcoal, alginate, bone powder, calcium carbonate,cellulose, chitin, clay minerals (e.g., bentonite), dolomite, humus,insoluble phosphates (e.g., rock phosphate), peat, soil, talc, titaniumdioxide, nut shells, crab shells, and lignins.

[0151] A solid biocontrol composition of the invention can be preparedby suspending a solid carrier in a culture or suspension of live orinactivated B. casidae, or a cell-free filtrate or cell fraction of a B.casidae culture or suspension, or a solution of an antimicrobialpreparation of the invention; and drying the suspension under mildconditions, such as evaporation at room temperature or vacuumevaporation at 65° C. or lower. Preferred carriers for use in preparingsolid biocontrol compositions are those which are not phytotoxic and,where live B. casidae is being carried, preferably non-bacteriocidal ornon-bacteriostatic.

[0152] To enhance binding or absorption of B. casidae cells and/or cyststo a carrier, an adhesive or binding agent known in art may be added tothe appropriate suspension prior to drying. Useful adhesives and bindingagents include, but are not limited to, agar, gelatin, sugars, syntheticglue, and vegetable glue (e.g., gum arabic). The dried material isfinely ground (i.e., at least 90% passing through 300 mesh) to produce asolid biocontrol composition.

[0153] In preferred embodiments, the solid biocontrol compositions areprepared with cultures or suspensions of live or inactivated B. casidaestrain 2.2N.

[0154] A solid biocontrol composition of the invention also can beprepared by spray- or freeze-drying cultures, cells (e.g., cell pastes),cell-free culture-filtrates, or cell- or culture-fractions.

[0155] A biocontrol composition of the invention may comprisegel-encapsulated B. casidae cultures, cells, cysts, cell-free filtratesor cell fractions, or antimicrobial preparations of the invention. Suchgel-encapsulated materials can be prepared by mixing a gel-forming agent(e.g., gelatin, cellulose, or lignin) with a culture or suspension oflive or inactivated B. casidae, or a cell-free filtrate or cell fractionof a B. casidae culture or suspension, or a spray- or freeze-driedculture, cell, or cell fraction or in a solution of antimicrobialpreparations of the invention; and inducing gel formation of the agent.

[0156] 5.5. Methods for Prevention or Treatment of Microbial Diseases ofPlants

[0157] The present invention also provides methods for treating andpreventing microbial diseases of plants. The biocontrol methods of theinvention generally comprise treating subject plants with one dose orseveral doses (“a regime”) of a biocontrol composition of the invention.The methods of the invention may be administered prophylactically(prevention), before the appearance of any disease symptoms on subjectplants or therapeutically (treatment), after disease symptoms have beendetected.

[0158] The treatment with a biocontrol composition can be made directlyonto a plant. In one embodiment, the plant is sprayed with a liquidbiocontrol composition of the invention. Preferably, the liquidbiocontrol composition comprises a culture or suspension of strain 2.2N,or cell-free filtrate, or spray- or freeze-dried material, or cellfraction thereof. More preferably, the liquid biocontrol compositioncomprises a culture or suspension of live cysts of strain 2.2N. Inanother embodiment, the plant is dusted with a solid biocontrolcomposition of the invention. Preferably, the solid biocontrolcomposition comprises live or inactivated cysts of strain 2.2N. Thedirect treatments are particularly effective in controlling air-bornemicrobial diseases.

[0159] The treatment with a biocontrol composition also can be madeindirectly by application to the soil in the immediate vicinity of aplant or in the area in which a seed has been, or will be, planted. Inone embodiment, the soil at the target site is irrigated with an aliquotof a liquid biocontrol composition of the invention. Preferably, thebiocontrol composition comprises a culture or suspension of live strain2.2N. In another embodiment, a plant seed is coated with a solid or gelbiocontrol composition of the invention using methods known in the art(see, e.g., U.S. Pat. No. 4,798,723). Preferably, the solid or gelbiocontrol composition comprises live strain 2.2N. In yet anotherembodiment, a solid or gel biocontrol composition of the invention ismixed into the target site. Such indirect treatments are particularlyeffective in controlling soil-borne microbial pathogens.

[0160] The effectiveness of treatment with a biocontrol composition maybe determined by comparing the number of disease lesions appearing intreated and untreated plants that all have been infected with amicrobial pathogen of interest. As used herein, an “effective” dose orregime of treatment with a biocontrol composition against a particularmicrobial pathogen is one whose application achieves at least 20%,preferably at least 50%, more preferably at least 80%, and mostpreferably at least 90%, disease control against that pathogen, asassessed using the method described in Section 6.3. An effective dose orregime may be achieved by adjusting the level of active ingredient(s) inthe biocontrol composition and/or the amount or frequency of biocontrolcompositions applied.

[0161] The biocontrol methods of the inventions may be beneficially usedto prevent or treat a wide variety of microbial diseases of plants.Fungal diseases of plants that can be prevented, controlled orameliorated by the methods of the invention include, but are not limitedto, those caused by fungi in any of the following genera: Alternaria,Aspergillus, Botrytis, Cercospora, Cercosporidium, Erysiphe, Geotrichum,Mycosphaerella, Mucor, Penicillium, Phoma, Phytophthora, Plasmopora,Pseudopeziza, Puccinia, Pythium, Rhizoctonia, Rhizopus, Septoria,Sporothrix, Stemphylium, Trichophyton, and Verticillium. Bacterialdiseases of plants that can be controlled or ameliorated by the methodsof the invention include, but are not limited to, those caused bybacteria in any of the following genera: Pseudomonas, Erwinia andXanthomonas.

[0162] The methods of the invention may be beneficially applied topreventing or treating fungal and bacterial diseases of a wide varietyof agronomically important plants including, but not limited to: cropplants such as corn, soybean, wheat, rice, alfalfa, sorghum, peanut,tobacco, cotton, flax, safflower, oats, and canola; fruits andvegetables such as tomato, pepper, cucumber, lettuce, green beans, limabeans, peas, cantaloupe, musk melon, citrus fruits, grapes, and banana;and ornamentals and cut flowers such as geraniums, azaleas, roses,tulips, petunias, orchids, carnations, poinsettias, chrysanthemums; andconifers such as pine, yew, spruce. The present invention is morecompletely illustrated by the following non-limiting examples.

6. EXAMPLES

[0163] 6.1. Isolation and Identification of Burkholderia casidae

[0164] 6.1.1. Methods for Isolation of Predator Bacteria

[0165] 6.1.1.1. Method One

[0166] Ten gram samples of a Hagerstown silty clay loam soil (pH 6.1), aCazenovia silt loam soil (pH 7.0) collected in VAuburn, NY, a Dresdensilt loam soil (pH 6.7) collected in Evansville, Wis., and a Dresdensilt loam soil (pH 5.7) collected in Evansville, Wis. were placed insterile 1 oz screw capped bottles. Soils were adjusted to 65%moisture-holding, capacity and a suspension of Micrococcus luteus wasadded to yield 2.5×10⁷ M. luteus cells (as colony-forming units) pergram dry weight of soil. The M. luteus suspension was prepared bycollecting cells from a culture grown in 0.25% (w/v) Brain HeartInfusion Broth (BHIB) for 5 days at 27° C. Cells were washed three timesin sterile tap water and suspended in a volume of sterile tap waterequal to that of the culture. Bottles were incubated at 27° C. with capsloosened. After 1 and 2 weeks of incubation, M. luteus suspensions at aconcentration of 7×10⁶ cells per gram of dry soil were added to maintainthe moisture level.

[0167] Survival of the M. luteus cells in the incubated soil samples was11% after 1 week incubation and less than 0.1% at 3 weeks incubation.

[0168] After 3 weeks incubation, 0.1 ml of dilutions of the soil samplesin water were spread lightly over lawns of M. luteus cells. Lawns of M.luteus were prepared on BHIB agar containing 0.25% (w/v) Brain HeartInfusion Broth and 1.5% (w/v) agar. Plates were inoculated with 0.2 mlof a water suspension of M. luteus cells prepared from a culture grownin 0.25% (w/v) BHIB for 5 days at 27° C. Plates were incubated 3 days at27° C. to allow sufficient growth of M. luteus and then spread withdilutions of each soil sample.

[0169] Predator bacteria were identified as colonies surrounded bycleared zones, free from cells of M. luteus. Such colonies were isolatedand characterized for predator activity.

[0170] Lysis of M. luteus cells was observed when suspensions of apredator bacteria were streaked perpendicularly across streaks of M.luteus on BHIB agar. At the point of intersection, the growth ofpredator bacteria moved progressively to the side of, and tracked along,the M. luteus streak. As a consequence, the M. luteus streak was coveredwith the growth of the predator bacteria and cells of M. luteus werelysed.

[0171] 6.1.1.2. Method Two

[0172] Predator bacteria were isolated employing soil column slides.Washed cells of M. luteus, prepared as described in Section 6.1.1.1.,were smeared on the surface of sterile glass slides. A glass ring 11 mmhigh by 25 mm in diameter was placed on the smear and the ring filledwith soil. The soils were those described in Section 6.1.1.1. The soilwas tamped lightly to ensure contact with the M. luteus smear.Moisture-holding capacity was adjusted to 65%, and incubation was at 27°C. in sterile petri dishes.

[0173] At the completion of incubation, excess soil was gently removedfrom the soil-column slides. For isolation of predator bacteria, thesoil was gently removed to expose the slide surface where the M. luteuscells had been smeared. An inoculating loop that had been heated andplunged hot into agar was touched against this area and then streakedthrough a lawn of M. luteus prepared as described in Section 6.1.1.1.

[0174] When material from the soil column slides was streaked throughthe M. luteus lawn, colonies of predator bacteria appeared as those thatcaused lysis of M. luteus in their immediate vicinity.

[0175] 6.1.1.3. Method Three

[0176] One-gram samples of a Hagerstown silty clay loam (pH 6.2),designated RS89, were placed in 18×150 mm screw cap tubes that alsocontained 10 ml of distilled water. The soil samples were sieved beforeuse with a 3 mm pore-size sieve and 0.8 mg of L-glutamic acid (SigmaChemical Co., St. Louis, Mo.) per gram of soil was added to the 10 mlwater. The soil suspension in the tube was mixed thoroughly with avortex mixer and then incubated with agitation for 24 hours at 28° C.

[0177] After incubation, the 10 ml of soil suspension plus 90 ml ofsterile distilled water (i.e., 100-fold dilution of soil) wastransferred to a sterile Waring blender head and blended 1 min. Further10-fold dilutions were prepared from this blended 100-fold dilution.Soil dilutions were plated on Copper medium agar containing (0.25% (w/v)BHIB, 0.01% (W/V) CuCl₂.2 H₂O and 1.5% agar (pH 6.5)).

[0178] Colonies of different morphologies appeared on Copper medium agarafter 1 to 7 days incubation at 28° C. B. casidae colonies were white,opaque, raised, beehive-shaped, and about 2 to 4 mm in diameter.Predator bacteria, including B. casidae, could be demonstrated asfollows. Putative predator bacteria were streaked near one edge of BHIBagar plates, and the plates were incubated for 24 hr at 28° C. A secondstreak of Saccharomyces cerevisiae or M. luteus was then inoculated onthe plates as a streak perpendicular to, but not quite touching, thepredator bacteria streak. The plates were then further incubated at 28°C. Predator bacteria were identified as those that produced zones ofcleaning of S. cerevisiae or M. luteus up to 20 mm in width.

[0179] 6.1.1.4. Method Four

[0180] Predator bacteria were isolated by plating dilutions ofunfiltered percolation fluid from an M. luteus-treated soil column onMacConkey agar (Difco, Detroit, Mich.). Percolation fluid was collectedfrom soil columns in the following manner. Twenty-five grams of soil(Table 1, infra) and 25 grams of sand were thoroughly mixed and placedin a soil percolation column in steps. A porous, plastic membranesupport from a membrane filter apparatus was used to support thesoil-sand mixture. A suspension of prey microorganism (e.g., M. luteus)was also added over and through the soil-sand column in steps. In step1, a 10 ml portion of the suspension was poured over about one-third ofthe soil-sand mixture. In the second step, the second third of thesoil-sand mixture was layered and 10 ml more of the cell suspension usedto wet the soil. This procedure was repeated for the final third of thesoil-sand mixture resulting in “saturation” of the soil-sand mixturewith prey microorganism.

[0181] The inoculated soil column was incubated at 27° C. At varioustime intervals (usually 24 hr), 10 ml of sterile distilled water wasadded to the top of the column and samples of percolation fluidcollected. Percolation fluid samples were spread, either undiluted ordiluted, on the surface of MacConkey agar. M. luteus fails to grow onthis medium and predator bacteria can be isolated and tested forpredator activity as described in Section 6.1.1.1. or 6.1.1.2. andpredator bacteria identified.

[0182] The soils that yielded predator bacteria are listed in Table 1below. TABLE 1 Soils Yielding Predator Bacteria. Soil Location Site pHHagerstown Silty Clay Loam State College PA Grass 6.1 Cazenovia SiltLoam Auburn NY Garden 7.0 Dresden Silt Loam Evansville WI Garden 6.7Webster Silty Clay Loam Monona IA Corn 5.6 Hagerstown Silty Clay LoamState College PA Grass 5.3 Tuxford Clay Loam Weyburn SASK Plain 7.2Yorkton Loam Watson SASK Plain 6.9 Waitville Loam Glaslyn SASK Plain 6.7

[0183] 6.1.1.5. Method Five

[0184] A soil-plating procedure was employed to isolate predatorbacteria. Plates were poured with 1.5% (w/v) Noble agar and allowed tosolidify. To the surface of the agar was added 0.2 ml of an Arthrobacterglobiformis (ATCC 8010) culture filtrate and 0.1 ml of a dilution insterile tap water of the soil sample. The A. globiformis culturefiltrate was prepared by inoculating BHIB and incubating with shakingfor 3 days at 30° C. The pH of the broth increased to 8.7 duringincubation. The cells were removed by centrifugation, and thesupernatant culture medium was sterilized by filtration through amembrane filter of pore size 0.22 μm.

[0185] After 7 to 11 days incubation of the plates at 30° C., the plateswere inspected for the presence of colonies having a “fried-egg” or“beehive” appearance. Detection and identification of such coloniesrequired 20-fold magnification because of their small size (i.e., 0.1 to0.5 mm diameter). These colonies were tested for predator activity asdescribed in Section 6.1.1.1. or 6.1.1.2 and predator bacteriaidentified.

[0186] 6.1.1.6. Method Six

[0187] The following soil-plating technique was also successfully usedto isolate predator bacteria. In this method, a soil dilution was spreaddirectly onto the surface of a modified Burk Azotobacter (BA) agar thatlacks a carbon source and contains only a minimal nitrogen level. The BAagar medium comprised 0.08% K₂HPO₄, 0.02% KH₂PO4, 0.02% MgSO₄.7H₂O,0.01% CaSO₄.2H₂O, 0.000025% NaMoO₄.2H₂O, 0.00086% Fe(NH₄)₂(SO₄)₂.12H₂O,and 1.5% Noble agar.

[0188] After 7 to 11 days incubation of the plates at 30° C., the plateswere inspected for the presence of colonies having a “fried-egg” or“beehive” appearance. Detection and identification of colonies required20-fold magnification because of their small size (i.e., 0.1 to 0.5 mmdiameter).

[0189] Colonies were tested for predator activity as described inSection 6.1.1.1. or 6.1.1.2. and predator bacteria identified.

[0190] 6.1.1.7. Method Seven

[0191] Predator bacteria also were isolated by a “baiting” techniquethat appears to operate on the basis of chemotaxis. Air-dried soil waspassed through a 1.19 mm sieve, then placed to a depth of 3-4 mm in asterile Petri dish (90 mm internal diameter). The soil was tampedlightly with the edge of a microscope slide to give a uniformly flatsurface. About 2 ml of sterile distilled water was then added along thewalls of the plate so the soil became moist but not flooded.

[0192] In a separate, sterile Petri dish, a 47 mm diameter membranefilter of 0.65 μm pore size was placed on a 8.5 cm circle of sterileWhatman No. 2 filter paper. To the membrane filter was added 0.2 ml of awashed suspension of M. luteus cells, prepared as described in Section6.1.1.1. The suspension was spread over a 28 mm diameter area in thecenter of the filter.

[0193] The membrane filter was then transferred onto the soil surfacewith the side having the cells facing the soil and pressed onto the soilsurface using a sterilized edge of a microscope slide. Each soil-filledPetri dish could hold 2 filters. The lids of the Petri dishes wereplaced on the filter-soil combination and plates sealed with tape andincubated right side up at 27° C. Cleared areas appeared on the filterswithin 2 to 3 days of incubation.

[0194] At various periods of incubation from 1 to 14 days, material fromcleared areas on the filters (e.g., filter and bacteria) were removedwith a sterile spatula and suspended in 5 ml sterile tap water. Aftermixing, a 2-fold dilution series was prepared in sterile tap water and0.1 ml of the undiluted and diluted suspensions were spread on thesurface of agar medium along with 0.1 ml of the washed M. luteussuspension prepared as described in Section 6.1.1.1. The media used were1.5% (w/v) Noble agar or BHIB agar.

[0195] The plates were incubated 3 days at 27° C. and inspected for theappearance of colonies surrounded by cleared zones of M. luteus cells.Such predator colonies were picked and purified on BHIB agar, to ensurethat non-obligate predator bacteria were recovered. Isolates were thentested for predator activity as described in Section 6.1.1.1. or6.1.1.2. and predator bacteria identified.

[0196] 6.1.2. Identification and Characterization of Strain 2.2N

[0197] 6.1.2.1. THE 16S rRNA Gene of Strain 2.2N

[0198] A culture of strain 2.2N was grown in 10 ml TSM+S medium in a 125ml Erlenmeyer flask with cotton stopper. The culture was inoculated witha single colony of strain 2.2N and incubated at 30° C. for 24 hours. Theculture was aerated by rotation at 60 rpm. Following incubation, cellswere harvested by centrifugation (5,000×G for 30 min at 4° C.). GenomicDNA was isolated using the Marmur method (i.e., Marmur, J. Mol. Biol.3:208-218, 1961).

[0199] The 16S rRNA gene was amplified using PCR employing universalbacterial primers for the 16S rRNA gene. Two sets of primers were usedto sequence the entire 16S rRNA gene.

[0200] The forward primers were:

[0201] 27f: 5′-AGA GTT TGA TCC TGG CTC AG-3′ (SEQ ID NO: 2)

[0202] 704f: 5′-GTA GCG GTG AAA TGC GTA GA-3′ (SEQ ID NO: 4)

[0203] The reverse primers were:

[0204] 1522r: 5′-AAG GAG GTG ATC CA (AG) CCG CA-3′ (SEQ ID NO: 3)

[0205] 907r: 5′-CCG TCA ATT CCT TTG AGT TT-3′ (SEQ ID NO: 5)

[0206] The PCR amplification was carried out using one microgram ofstrain 2.2N DNA with 3.2 pmol each of a forward and a reverse primer.

[0207] The sequence of the amplified product was determined using an ABIPrism TM377 automated DNA sequencer following the instructions providedby the manufacturer. Taq DNA polymerase was used in DyeDeoxy™ terminatorcycle sequencing reactions. All four termination reactions wereperformed in a single reaction and the products were loaded into asingle lane of a polyacrylamide gel for separation. Real time detectionof separated, individual fragments was achieved with laser scanning.

[0208]B. casidae appears to have five 16S rRNA operons. The DNAsequenced was a PCR product of genomic DNA generated by primers 27f and1522r. The sequence covers position 27 through position 1522 (thenumbers reflect the numbering of the Escherichia coli 16S rRNA gene) ofone, and possibly all five, 16S rRNA genes. Thus, the sequence shown inFIG. 1 covers greater than 98% of 16S rRNA sequence (see FIG. 1; SEQ IDNO: 1). A sequence alignment program produced by DNASTAR, Inc.,(Madison, Wis.) was employed to analyze the 16S rRNA sequence and toresolve any ambiguities or errors in sequencing.

[0209] A general description of the amplified 16S rRNA gene sequence ofstrain 2.2N is summarized in Table 2, below. TABLE 2 General Descriptionof the amplified 16S rRNA Gene Sequence of Strain 2.2N Total number ofbases is 1495 % A = 25.35 % G = 31.57 % T = 20.13 % C = 22.94 %Ambiguous 0.00 % A + T = 45.48

[0210] Pairwise comparisons of 16S rRNA gene sequences between that ofstrain 2.2N and those of other bacteria were performed by “ClustalAnalysis” employing DNASTAR. The results of the pairwise comparison ofsimilarities of 16S rRNA gene sequences of strain 2.2N with that ofother bacteria, particularly members of the genus Burkholderia, arepresented in Table 3. TABLE 3 Percent Similarity of Paired 16S rRNA GeneSequences of Members of the Genus Burkholderia and Escherichia ColiBacterial Strains^(a) 2.2N 23061 25418 x80284 10248 s55000 R780 X807242.2N 100 88.8 93.0 94.6 94.5 94.1 86.3 71.7 23061 88.8 100 93.2 92.294.7 93.6 88.3 76.0 25418 93.0 93.2 100 93.2 95.3 93.7 87.8 75.4 X8028494.6 92.2 93.2 100 95.4 90.7 87.7 74.1 10248 94.5 94.7 95.3 95.4 10095.5 88.8 75.2 S55000 94.1 93.6 93.7 90.7 95.5 100 89.1 74.3 R780 86.399.3 87.8 87.7 88.8 89.1 100 73.5 X80724 71.7 76.0 75.4 74.1 75.2 74.373.5 100

[0211] The high degree of similarity of the 16S rRNA sequence of strain2.2N with representatives of Burkholderia species indicates that strain2.2N is a member of the genus Burkholderia.

[0212] 6.1.2.2. Fatty Acid Composition of Strain 2.2N

[0213] A culture of strain 2.2N was streaked on TSM agar and incubatedat 30° C. After 24 hour incubation, approximately 40 mg of cell materialwas harvested and cellular fatty acids extracted and transformed tomethyl esters for gas chromatography (GC) analysis. Specifically, cellmaterial was first saponified, then the cellular fatty acids weremethylated, and then the methyl esters of the fatty acids extracted.Extracted fatty acid methyl esters of 9 to 20 carbons in length wereidentified and their percent composition was determined by GC analysis.Individual peaks were identified by retention time in comparison tothose of a library of fatty acids, and the area under each peakmeasured.

[0214] The individual cellular fatty acids and percent composition ofeach, for strain 2.2N, are listed in Table 4. TABLE 4 Cellular FattyAcid Composition of Burkholderia spp. and Burkholderia casidae strain2.2N Fatty Acid A B C D E F G H I C10:0 1 — — — — — — — ND C12:0 4 — — —— — — — 0.8 C13:1 ND ND ND ND ND ND ND ND 0.7 C14:0 1 5 4 4 4 4 5 5 2.9C15:0 1 1 — 1 1 — 2 1 ND C16:0 36  29  22  26  28  31  34  25  18.1 C16:1 6 3 4 5 8 4 22  9 21.1  C16:17,8 ND ND ND ND ND ND ND ND ND C17:0— — 3 1 — — 1 — 0.4 C17:CPA 3 21  10  12  24  24  1 27  3.7 C18:0 1 8 —1 — 1 — 1 1.1 C18:1 (9,10) 2 — — 1 — — — — ND C18:1 (11,12) 22  8 20 18  12  8 18  13  39.2  C19:CPA 7 15  9 7 3 8 — — 1.5 2-OH C12:0 5 — — —— — — — 0.7 2-0H C15:0 ND ND ND ND ND ND ND ND ND 2-OH C16:0 — 3 6 4 2 21 1 0.5 2-OH C16:1 — 1 3 1 5 1 1 5 0.7 2-OH C18:1 — 1 3 2 4 2 4 4 1.12-OH C19:CPA — 2 2 2 — 2 — — ND 3-OH C10:0 3 — — — — — — — ND 3-OH C12:08 — — — — — — — ND 3-OH C14:0 — 4 6 6 9 5 9 4 4.2 3-OH C16:0 — 6 8 8 — 8— — 3.4

[0215] Table 4 also contains data for fatty acids and their percentcomposition in cells of Burkholderia cepacia, Burkholderia mallei,Burkholderia pseudomallei, Burkholderia caryophyli, Burkholderiagladoli, Burkholderia solanacearum, and Burkholderia pickettii. The datawere obtained from Yabuuchi et al., 1992, Microbiol. Immunol.36:1251-1275.

[0216] The cellular fatty acid profile of strain 2.2N differs fromrepresentatives of other Burkholderia species in having a significantlylower percentage of C16:0 fatty acid and significantly higherpercentages of C16:1 and C18:1 (11,12) fatty acids.

[0217] Since strain 2.2N has a unique cellular fatty acid profile, notdisplayed by representatives of any known species of Burkholderia, itis, therefore, a representative of a new Burkholderia species.

[0218] 6.1.2.3. Carbon Utilization by 2.2N

[0219] Fifty-four substrates were tested to identify those which strain2.2N uses as carbon and energy sources. Substrate utilization wasdetermined using the API50 CH test (bioMerieux S. A., Marcy-l'Etiole,France) following the instructions provided by the manufacturer. Theresults of that determination are listed in Table 5. TABLE 5 SubstrateUtilization Patterns of Burkholderia spp. and Strain 2.2N Burkholderiaspecies casidae Substrate cepacia marginata allicola caryophilasolanacearum pickettii gladioli mallei 2.2N 2-ketogluconate + + + +− + + − − 5-ketogluconate + ND ND + − − + ND − a-methyl-D-glucoside NDND ND ND ND ND ND ND − a-methyl-D-mannoside − ND ND − − − − − −adipate + + + − − + + + + adonitol + + + − − − + ND − amygdalin + ND ND− − − − ND − arbutin + ND ND − − − − ND − b-methyl-D-xyloside ND ND NDND ND ND ND ND ND caprate + + + − − − + − + cellobiose + + + + − * − + −citrate + + + + ND + ND ND − D-arabinose + + + + − − + + − D-arabitol +ND ND ND − − + + − D-fucose + + + + − − + + + D-lyxose + ND ND ND ND NDND ND ND D-tagatose + ND ND ND − − + ND − D-turanose ND ND ND ND − ND −ND − D-xylose + + + + − + + + − dulcitol + ND ND ND − − + ND −erythritol − − − − − − − − − esculin + ND ND − − − − − +fructose + + + + + + + + + galactose + + + + − + + + + gentiobiose ND NDND ND ND ND ND ND ND gluconate + + + + ND + ND ND + glucose + + + +− + + + + glycerol + + + + + + + + − glycogen − ND ND ND − − − ND −inositol + + + + − − + + − inulin − − − − − − − − − L-arabinose + + + +− + + − − L-arabitol + ND ND ND − − − ND − L-fucose + ND ND ND − − + ND− L-xylose ND ND ND ND − ND − ND − lactose − − − − − − − − −malate + + + + − + + − − maltose − − − − − − − − − mannitol + + + + −− + + + mannose + + + + − − + + + melezitose − ND ND ND − − − − −mellibose − ND ND ND − − − − + N-acetyl-glucosamine + ND ND ND + + + + +phenylacetate + + + + − − + + + raffinose − ND ND ND − − − − − rhamnose− − − + − − − − − ribose + ND ND ND − − + − − salicin + + + + − − − − −sorbitol + + + + − − + + − sorbose ND ND ND ND ND ND ND ND − starch − −− − − − − ND − sucrose + + + + + − − + + trehalose + + + − − − + + −xylitol + ND ND ND − − + ND −

[0220] Table 5 also lists substrates used as carbon and energy sourcesby strains of Burkholderia cepacia, Burkholderia marginata, Burkholderiaallicola, Burkholderia caryophyli, Burkholderia solanacearum,Burkholderia pickettii, Burkholderia gladoli, and Burkholderia mallei.The data were obtained from Ballard et al., 1970, J. Gen. Microbiol.60:199-214; Ralston et al., 1973, Int. J. System. Bacteriol. 23:15-19;Palleroni and Holmes, 1981, Int. J. System. Bacteriol. 31:479-481;Yabuuchi et al., 1992, Microbiol. Immunol. 36:1251-1275; and Bevivino etal., 1994, Microbiol. U.K. 140:1069-1077.

[0221] The pattern of carbon and energy sources utilized by strain 2.2Nis unique among members of the genus Burkholderia. Based on the lack ofany significant degree of similarity between the patterns of substrateutilization of other Burkholderia species and that of strain 2.2N, itwas concluded that strain 2.2N is a representative of a new Burkholderiaspecies.

[0222] 6.1.2.4. Cultural, Biochemical, and Enzymatic Characteristics ofStrain 2.2N

[0223] Cultural, biochemical, and enzymatic activities of strain 2.2Nwere determined using the API-NFT test (bioMerieux S. A.,Marcy-l'Etiole, France) following the instructions provided by themanufacturer.

[0224] The results of the API-NFT test for strain 2.2N are shown inTable 6. Table 6 also contains data for corresponding cultural,biochemical, and enzymatic activities of strains of Burkholderiacepacia, Burkholderia mallei, Burkholderia pseudomallei, Burkholderiacaryophyli, Burkholderia gladoli, Burkholderia pickettii, andBurkholderia solanacearum. The data were obtained from Ballard et al.,1970, J. Gen. Microbiol. 60:199-214, Ralston et al., 1973, Int. J.System. Bacteriol. 23:15-19; Palleroni and Holmes, 1981, Int. J. System.Bacteriol. 31:479-481; Yabuuchi et al., 1992, Microbiol. Immunol.36:1251-1275;, and Bevivino et al., 1994, Microbiol. U.K. 140:1069-1077.TABLE 6 Cultural, Biochemical, and Enzymatic Characteristics of B.casidae strain 2.2N and Burkholderia spp. Character A B C D E F G HGrowth at 41° C. + − + − − + − + Yellow Pigment + − − − − + − − NO₃ toNO₂ − + + + − + + + Urease − − − − − + + − Esculin Hydrolysis + − − − −− − + Gelatin Hydrolysis + − − − + + + + Arginine − + + − − − − −Dihydrolase Cytochrome − + + − + + + − Oxidase Indole Formation − − − −− − − +

[0225] Strain 2.2N displays a unique pattern of cultural, biochemical,and enzymatic activities that is not shown by any other members of thegenus Burkholderia. Based on the lack of any significant degree ofsimilarity between the patterns of cultural, biochemical, and enzymaticactivities of representatives of other Burkholderia species and that ofstrain 2.2N, it was concluded that strain 2.2N is a representative of anew species of Burkholderia.

[0226] 6.1.2.5. Antibiotic Susceptibility of Strain 2.2N

[0227] The susceptibility of strain 2.2N to various differentantibiotics was tested employing the Sceptor Pseudomonas/Resistant MICPanel (Becton-Dickinson, Sparks, Nev.) following the instructions of themanufacturer.

[0228] The susceptibility or resistance of strain 2.2N to antibiotics isdisplayed in Table 7. Strain 2.2N is resistant to most antibioticscommonly used to treat Pseudomonas spp. infections. TABLE 7 MinimalInhibitory Concentrations of Antibiotics Against B. casidae strain 2.2N.Minimal Inhibitory Antibiotic Concentration¹ Cefotaxime 64 micrograms/mlCeftizoxime 16 micrograms/ml Ceftazimine 8 micrograms/ml Ceftriaxone 128micrograms/ml Imipenem 16 micrograms/ml Trimethoprim/Sulfamethoxazole4/76 micrograms/ml Amikacin >64 micrograms/ml Cefoperazone >16micrograms/ml Gentamycin >16 micrograms/ml Tetracycline >8 micrograms/mlTricarcillin/Clavulinic Acid >128/2 micrograms/ml Tobramycin >16micrograms/ml

[0229] 6.2. Production and Assay or Antimicrobial Compounds Produced byBurkholderia casidae

[0230] 6.2.1. Growth of Strain 2.2N

[0231] A single colony of strain 2.2N was picked and used to inoculate10 ml of TSM+S medium. The inoculum culture was incubated 18 hours at30° C. and refrigerated. It could be stored for up to 2 weeks prior touse as inoculum.

[0232] Test cultures were always assayed immediately followingincubation and growth. However, antimicrobial compounds in the culturewere stable for over 2 months storage, in a refrigerator (4° C.) or atroom temperature, and their activity may be assayed at any time duringthat period.

[0233] 6.2.2. Fractionation of Strain 2.2N Culture

[0234] In addition to whole cultures designated as “culture”, andcell-free culture medium in which strain 2.2N had grown, antimicrobialpreparations were made from strain 2.2N culture, and all were assayedfor antimicrobial activity.

[0235] Production cultures of strain 2.2N to be used for dose-responsemeasurements consisted of either 250 ml of TSM+S medium in a screw-cap1-liter Erlenmeyer flask (flask culture) or 1,000 ml of TSM+S medium ina 2-liter fermenter (fermenter culture). Production cultures wereinoculated with a 1:100 dose of starter culture (e.g., 2.5 ml of starterculture per 250 ml of fresh medium) and incubated 24 hours at 30° C.with aeration by rotation (60 rpm).

[0236] Several different materials were prepared from the productionculture and tested for inhibitory activity against a number of fungi.

[0237] A fraction of a production culture was centrifuged (5,000×G for30 min at 4° C.) and the supernatant liquid collected. A portion of thesupernatant, spent medium was filtered through a 0.22 μ pore sizefilter. This material was designated “Filtrate.” A second portion of thesupernatant, spent medium was heated to 80° C. for 5 minutes. Thismaterial was designated “Pasteurized.”

[0238] The pellet from the centrifugation of culture was suspended in avolume of sterile distilled water equal to that of the original sampleand resuspended. That fraction as designated “Cells”.

[0239] Antimicrobial preparations were made from the production culturefollowing procedures described in Section 6.5.1., below. Thepreparations were dissolved in buffer. This material was designated“Fungicide.”

[0240] Another material tested was simply an untreated sample of theproduction culture. This material was designated “Culture.”

[0241] 6.2.3. Growth of Target Microorganisms

[0242] 6.2.3.1. Yeasts

[0243] Single colonies of S. cerevisiae, C. albicans, or C. neoformanswere picked and used to inoculate 20 ml of BHIM. The cultures wereincubated 18 hours at 30° C. with shaking. Inoculum cultures grown insuch a manner could be refrigerated for up to 2 weeks prior to use inactivity assay.

[0244] A single colony of S. cerevisiae was picked and used to inoculate10 ml of BHIM. The inoculum culture was incubated 18 hours at 30° C.with shaking and refrigerated. It could be stored for up to 2 weeksbefore use in assay.

[0245] 6.2.3.2. Micrococcus luteus

[0246] A single colony of M. luteus was picked and used to inoculate 10ml of BHIM. The culture was incubated 18 hours at 30° C. with shakingand refrigerated. It could be stored for up to 2 weeks prior to use inactivity assay.

[0247] 6.2.3.3. Staphylococcus aureus

[0248] A single colony of S. aureus was picked and used to inoculate 10ml of BHIM. The inoculum culture was incubated 18 hours at 30° C. andrefrigerated. It could be stored for up to 2 weeks prior to use inactivity assay.

[0249] 6.2.3.4. Aspergillus niger

[0250] A single colony of A. niger was picked and used to inoculate thesurface of solid medium consisting of BHIM agar. The plate was incubatedat 30° C. for 5 days, or until spore formation was evident from theblack spores. Spores were harvested from the agar by adding 5 ml ofwater to the agar surface of the medium and gently dislodging the sporesby repeatedly passing the side of a sterile glass rod over the surface.The spore-laden liquid was collected using a pipette and suspensions ofsingle spores prepared by, first, a low speed centrifugation (1,000×Gfor 5 min at room temperature) to remove pelleted debris and, second, bya high speed centrifugation (5,000×G for 30 min at room temperature).The pelleted spores were collected, washed twice in distilled water, andresuspended in medium prior to use in activity assay.

[0251] 6.2.3.5. Botrytis cinerea

[0252] A single colony of B. cinerea was picked and used to inoculatethe surface of solid medium consisting of 20% (v/v) V8 Juice™ containing0.25% (w/v) CaCO₃ and 1.5% (w/v) agar. The plate was incubated at roomtemperature for 5 days, or until spore formation was evident from thegray spores. Spores were harvested and prepared as described above forcollection of A. niger spores.

[0253] 6.2.3.6. Septoria nodorum

[0254] A single colony of S. nodorum was picked and used to inoculatethe surface of solid medium consisting of 20% (v/v) V8 Juice™ containing0.25% (w/v) CaCO₃ and 1.5% (w/v) agar. The plate was incubated at roomtemperature for 5 days, or until spore formation was evident from thegray spores. Spores were harvested and prepared for use in activityassay following procedures described above for collection of A. nigerspores.

[0255] 6.2.4. Activity Assay

[0256] One-tenth strength Brain Heart Infusion Broth containing 0.7%(w/v) agar, designated BHIM top agar, was prepared and sterilized byautoclaving and dispensed into 3 ml volumes in 13×100 mm tubes andcooled to 45° C. One-tenth ml of an inoculum culture or spore suspensionof each prey microorganism was added to 3 ml of 45° C. BHIM top agar,mixed and poured over the surface of agar medium made up of BHIM or amedium composed of 20% (v/v) V8 Juice™ 0.25% CaCO₃ and 1.5% (w/v) agar.The later agar medium is used for activity for assays using B. cinereaor S. nodorum as prey microorganism. Immediately, or within 1 hour, 10μl samples of the test material (see Section 6.2.2.) were spotted on thesurface of the test plates and allowed to air dry. Plates were incubatedat 30° C. and examined at hourly intervals for the appearance of zonesof inhibition in the lawns of prey microorganism surrounding the spotsof the test material. As control, 10 μl of sterile TSM was spotted oneach lawn. Inhibitory activity of the test materials was assessed bymeasuring the diameter (mm) of the zones of inhibition of prey growthsurrounding the spots of the test material.

[0257] 6.2.5. Results

[0258] There was no inhibition of the prey microorganisms where 10 μl ofthe sterile TSM (control) had been spotted. Though there was growth ofstrain 2.2N cells in the centers of the zone of inhibition of spottedcultures or cells, there was no growth of cells in the zones ofinhibition of the “Filtrate” or the “Pasteurized” materials. Theinhibitory activity of the different types of sample material are shownin Tables 8 and 9, below. TABLE 8 Inhibitory activity of strain 2.2NCulture, Filtrate and Cells. Zone of Inhibition (mm ± std. dev.) No. ofNo. of Sample Trials S. cerevisiae Trials M. luteus Culture 16.8 ± 1.2 117.0 Filtrate 8  9.9 ± 3.1 1 13.0 Cells 8 14.0 ± 2.3 1 17.0

[0259] TABLE 9 Inhibitory Activity of Materials Prepared From Strain2.2N Culture. Culture Pasteurized Filtrate Fungicide S. cerevisae 17 ±3.1^(a) 11 ± 3.1 (57) 10 ± 3.2 (18) 13 ± 3.8 (49) (519) C. albicans 15 ±2.6 (12) 10 ± 1.0 (4)  8 ± 0.0 (1)  8 ± 3.3 (5) C. neoformans 20 ± 3.5(16) 11 ± 1.0 (3)  8 ± 0.7 (2)  9 ± 2.8 (5) A. niger 22 ± 4.8 (36) 12 ±5.0 (5) 12 ± 0.7 (2) 13 ± 5.3 (42) B. cinerea 37 ± 0.0 (2) ND^(b) ND 13± 3.5 (2) S. nodorum 27 ± 6.2 (5) ND ND 22 ± 1.4 (2) M. luteus 18 ± 3.0 9 ± 3.3 (4) 12 ± 4.7 (6) 14 ± 5.7 (9) (400) M. smegmatis 21 ± 3.4 (13)ND ND 13 ± 0.7 (2)

[0260] 6.2.6. Effect of Growth Conditions on Production of AntimicrobialCompounds

[0261] The effect of different media conditions and growth conditions onthe production of antimicrobial activity was examined. Strain 2.2N wasgrown using conditions described in Table 10. The inhibitory activity of2.2N cultures grown under the different conditions was assayed asdescribed in Section 6.2.4.

[0262] The results, as shown in Table 10, indicated that cultures grownat 37° C., or anaerobically, produced lower levels of anti-fungal andantibacterial compounds, and that cultures grown in medium supplementedwith ammonium or sulfate produced relatively more antibacterial thananti-fungal compounds. TABLE 10 Influence of Medium and CultureConditions Production of Antimicrobial Compounds by Strain 2.2N. CultureNo. of Diameter (mm) of Zone of Inhibition Medium^(a) Trials S.cerevisiae M. luteus S. aureus Standard 6 18.5 18.5 10.0 Std + NH4 118.0 22.0 N.T. Std + SO4 1 18.0 21.0 N.T. Std + EDTA 1 17.0 17.0 N.T.Std + Metals 1 18.0 19.0 N.T. Std + PO4 1 18.0 18.0 12.0 Std − Buffer 118.0 18.0 11.0 Std + Tris 1 18.0 18.0 13.0 Std @ 37° C. 1 11.0 17.0 N.T.Std − Air 1 16.0 16.0 N.T.

[0263] 6.3. Biocontrol Activity of Burkholderia casidae

[0264] 6.3.1. Growth of Strain 2.2N

[0265] Production culture of strain 2.2N was prepared as described inSection 6.2.1., above.

[0266] 6.3.2. Biocontrol Activity Assay

[0267] Biocontrol activity of strain 2.2N was assessed by placingfourteen plants (two to three plants per treatment) in a tray, andapplying diluted or undiluted production culture of strain 2.2N with ahand-held sprayer. The activity of antimicrobial preparations preparedas described in Section 6.5. was also assayed. This material wasdesignated as “Fungicide”. Doses of the Culture and Fungicide testedincluded: undiluted (1×), one-third diluted (⅓×), one-tenth diluted({fraction (1/10)}×), one-thirtieth diluted ({fraction (1/30)}×),one-hundredth diluted ({fraction (1/100)}×), one-three-hundredth diluted({fraction (1/300)}×), and on-one-thousandth diluted ({fraction(1/1,000)}×). Plants were sprayed with each of these preparations untilthoroughly wet. These are referred to as “treated” plants. Severalplants were not sprayed. They served as untreated disease controls.Following spraying, plants were placed in separate trays, one for eachfungal disease and allowed to air-dry for one to two hours.

[0268] Biocontrol activity of the Culture and Fungicide were testedagainst various fungal diseases of wheat, tomato, grape, rice, banana,peanut and pepper. Wheat plants were inoculated with Erysiphe graminisby shaking infected plants containing sporulating lesions over trays ofstrain 2.2N treated and untreated (control) plants. All other fungalinoculations were carried out by spraying the plants with a suspensionof fungal spores.

[0269] The spore inoculated plants were placed in humidity chambers setat temperatures conducive to the development of each fungal disease. Inaddition to the untreated control plants that were inoculated withfungal spores, plants that were both untreated and uninoculated wereincluded to provide disease-free controls.

[0270] All plants (i.e., uninoculated, inoculated, and inoculated andtreated) were evaluated by visually estimating the level of diseasecontrol. The plants inoculated with fungi only were assigned a rating of0%. Untreated and uninoculated plants were assigned a rating of 100%.These were used as comparison standards. Plants showing no diseaselesions were given a rating of 100%. Plants with intermediate levels ofdisease were assigned appropriate intermediate values between 0 and100%. Replicate plants were visually averaged and one disease rating wasassigned for each treatment for each disease. Uninoculated plants thatwere treated with 2.2N were also observed for effects of the treatmentsother than disease control (e.g., phytotoxicity).

[0271] These assays also compared the activity of the Culture andFungicide preparations (see above) in controlling Botrytis cinereainfection of geranium with that of a chemical fungicide Rovral® 50 WP(iprodine). The Rovral® was dissolved to 1 lb/100 gal in an aqueoussolution of 5% acetone, 0.25% Triton X-155, and applied by spraying asdescribed above for 2.2N Culture and Fungicide preparations.

[0272] 6.3.3. Results of Biocontrol Activity Assays

[0273] The results shown in Table 11 (below) list the average (±standard deviation) disease control activity of undiluted productioncultures of strain 2.2N. Tomato, grape, pepper and wheat plants treatedwith strain 2.2N and not inoculated with pathogens showed no disease orphytotoxic symptoms. TABLE 11 Biocontrol Activity of Strain 2.2N PercentDisease Fungus Plant Trials Control Phytophthora infestans Tomato 8  70± 19 Alternaria solani Tomato 8  64 ± 28 Plasmopora viticola Grape 8  60± 42 Botrytis cinerea Pepper 8 100 ± 0 Septoria nodorum Wheat 8  96 ± 7Puccinia recondita Wheat 8  63 ± 42

[0274] Tables 12 and 13, below, show the activity of undiluted anddiluted 2.2N Culture and Fungicide preparations in controlling variousfungal diseases of tomato, rice, pepper, wheat, banana and peanut. Theresults show that the 2.2N Culture and Fungicide preparations can bediluted 3-fold and still exert good biocontrol activity against fungalinfections caused by Phytophthora infestans, Pyricularia oryzae,Botrytis cinerea, Septoria nodorum, Puccinia recondita, Septoriatritici, Mycospharella fijiensis, Cerospora arachidocola, andCercosporidium personatum. TABLE 12 Antifungal activity of Culture andFungicide preparations. Percent Disease Control Culture Fungicide FungusHost Plant 1X 0.33X 1X 0.33X Phytophthora tomato 90 70 53 23 infestansPyricularia rice 100 50 93 17 oryzae Botrytis cinerea pepper 100 100 10053 Septoria nodorum wheat 100 99 92 63 Puccinia wheat 100 99 96 87recondita

[0275] TABLE 13 Antifungal activity of Culture and Fungicidepreparations. Percent Disease Control Culture Fungicide Fungus HostPlant 1X 0.33X 1X 0.33X Septoria tritici wheat 70 58 75 63 Mycospharellabanana 100 91 100 100 fijiensis Cerospora peanut 91 84 92 85arachidocola Cercosporidium peanut 91 84 92 85 personatum

[0276] Comparisons with the chemical fungicide Rovral® shows that boththe 2.2N Culture and Fungicide preparations have good activity incontrolling B. cinera infection of geranium (Table 14). TABLE 14 Percentcontrol of B. cinerea infection of geraniums treated with strain 2.2NCulture and Fungicide preparations and Rovral ®. Percent Disease Controldilution X 1/3X 1/10X 1/30X 1/100X Culture 85 75 15  6 0 Fungicide 86 8573 25 0 concentration 600 ppm 200 ppm 60 ppm 20 ppm 6 ppm Rovral ® 96 8135  0 0

[0277] The results of Table 15, below, show that production cultures ofstrain 2.2N can be diluted 10 to 30 fold and still exert significantbiocontrol activity against plant fungal pathogens. TABLE 15 Titrationof Inhibitory Activity in 2.2N Cultures Percent Disease Control Dilutionof Pepper Wheat Production inoculated with inoculated with TreatmentCulture B. cinerea S. nodorum Flask 1 X 92 99 Culture 1/3 X 92 94 1/10 X80 74 1/30 X 76 49 1/100 X 61 22 1/300 X 9 4 1/1,000 X 4 0 Fermentor 1 X93 100 Culture 1/3 X 75 97 1/10 X 75 85 1/30 X 69 60 1/100 X 64 37 1/300X 29 22 1/1,000 X 4 10

[0278] 6.4. Biocontrol Activity of Spray-Dried Cells of Burkholderiacasidae

[0279] 6.4.1. Growth of Strain 2.2 N

[0280] Production culture of strain 2.2 N was prepared as described inSection 6.2.1., above.

[0281] 6.4.2. Preparation of Cell Paste

[0282] The culture was passed through a Sharples centrifuge and thecells harvested from the walls of the chamber. The resulting cell pastehad approximately 32% solids and a pH of 4.5. The mean particle size was0.9 microns.

[0283] 6.4.3. Spray Drying

[0284] The cell paste, prepared as described in section 6.4.2, wasdiluted to about 28.5% solids and spray dried at a dried inlettemperature of 170° C. and outlet temperature of about 100° C. The flowrate of the feedstock (i.e., cell paste) was approximately 85 ml perminute. The main chamber yield of the dry product from the feedstock wasabout 21% and from the cyclone about 28% (total recovery equal to about49% of feedstock). The main chamber and cyclone collected products werecombined for physical and anti-microbial assays. The dried productmoisture content was about 5% and the particle size mean was about 0.85microns.

[0285] 6.4.4. Biocontrol Activity Assay

[0286] One gram of the spray-dried powder was dissolved in 100 ml ofwater (1×concentration). Doses of the spray-dried Material testedincluded: undiluted (1 gm in 100 ml=1×), one-tenth diluted ({fraction(1/10)}×), one-hundredth diluted ({fraction (1/100)}×), andone-three-hundredth diluted ({fraction (1/300)}×). Biocontrol activityof the spray-dried material and dilutions were assessed as described inSection 6.3.2., above.

[0287] 35 6.4.5. Results of Biocontrol Activity Assays

[0288] The results of measurement of the anti-fungal activity of thespray-dried material produced from cultures of B. casidae strain 2.2 Nare displayed in Table 16 (below). Table 12 lists the percent diseasecontrol of the undiluted and diluted spray-dried material and of theculture used to prepare the spray-dried material. TABLE 16 BiocontrolActivity of Spray-Dried Cells of B. casidae strain 2.2N Disease Control(%) Formulation Sample Pi Po Bc Sn Pr Spray Dried 1 X 95 90 100 100 1001/10 X 80 75 100 90 65 1/100 X 75 0 100 0 0 1/300 X 0 0 0 0 0

[0289] The results show that the spray-dried material prepared fromcells harvested from a culture of B. casidae strain 2.2 N can be dilutedup to 100-fold and still exert complete biocontrol activity againstfungal infection caused by Botrytis cinerea and 75% biocontrol activityagainst fungal infection caused by Phytophthora infestans. Control offungal infections was also provided by the 10-fold diluted productagainst fungal infections caused by Pyricularia oryzae (75% protection),Septoria nodorum (90% protection), and Puccinia recondita (65%protection).

[0290] 6.5. Production and Activity of Antimicrobial Preparations ofBurkholderia casidae Strain 2.2N

[0291] 6.5.1. Production of Antimicrobial Preparations

[0292] A culture of Burkholderia casidae strain 2.2N, grown as describedabove (Section 6.2.1), was centrifuged (5,000×G for 30 min at 4° C.),and the supernatant liquid collected.

[0293] Two volumes of 70% (v/v) isopropanol were added to thesupernatant and the resultant solution boiled for 1 minute. The solutionwas cooled to room temperature, then centrifuged (5,000×G for 30 min atroom temperature), and the supernatant liquid collected. The solutionwas evaporated to the original culture volume in a rotary vacuumevaporator at 65° C. Talc (magnesium silicate) was added to the liquidat a ratio of 4 grams talc per 50 ml liquid. The suspension was stirredat room temperature for 30 min. The talc fraction was separated from theliquid by centrifugation (5,000×G for 30 min at room temperature). Thetalc fraction was collected and washed twice with distilled wateremploying centrifugation (5,000×G for 30 min at room temperature). Theretained talc fraction was extracted with a 10-fold excess volume of 70%(v/v) isopropanol. The isopropanol-fraction, containing the elutedantimicrobial activity, was recovered by centrifugation (5,000×G for 30min at room temperature). The isopropanol fraction was evaporated todryness in a rotary vacuum evaporator at 65° C. and the antimicrobialpreparations weighed. The antimicrobial preparations was dissolved inwater to a concentration of 1 mg dry weight per ml prior to use inactivity assays.

[0294] 6.5.2. Activity of Antimicrobial Preparations

[0295] Antifungal activity of the antimicrobial preparation was assayedusing procedures described in Section 6.2., above.

[0296] Ten microliter volumes of test material (cultures orantimicrobial preparations) were spotted on test plates overlaid withprey microorganisms and allowed to dry. Plates were incubated at roomtemperature and examined at hourly intervals for the appearance of zonesof inhibition of microbial growth. Activity was measured as the diameter(mm) of the zones of inhibition of fungal growth.

[0297] As shown in Table 9, above, the antimicrobial preparation hasantifungal activity.

[0298] 6.6. Thermal Stability of Biocontrol Compositions

[0299] This section presents data on the thermostability of the variousdifferent inhibitory activities present in biocontrol compositions ofthe invention. A strain 2.2N culture was grown as described in Section6.2.1 and used to prepare “Culture”, “Filtrate” and “Fungicide”preparations as described in Section 6.2.2. Further, a portion of thesupernatant, spent medium was treated with −70° C. acetone to finalconcentration of about 70% (v/v). The precipitated material wascollected by centrifugation, dried, and dissolved in 8% (w/v) NaCl. Thisfraction was shown to have protease activity, and thus was designatedthe “Protease” preparation.

[0300] Each of these different preparations was treated for 15 min attemperatures ranging from 30° C. to 121° C. The biocontrol activity ofuntreated and heat-treated preparations was tested against Saccharomycescerevisiae (anti-yeast activity), Micrococcus luteus and Escherichiacoli (antibacterial activity), and Aspergillus niger, Botrytis cinerea,and Septoria nodorum (anti-filamentous fungi activity) following theprocedures described in Section 6.2.4.

[0301] Table 17 shows that the “Protease” preparation, like the Culturepreparation, contains at least three different biocontrol activities:anti-bacterial, anti-yeast and anti-filamentous fungi. TABLE 17Biocontrol Activity of Protease Preparation. Dia. of Zone of InhibitionMicroorganism Culture Protease Saccharomyces 20 mm 17 mm cerevisiaeMicrococcus luteus 23 mm 12 mm Aspergillus niger 27 mm 16 mm Escherichiacoli ND¹ 14 mm Septoria nodorum 22 mm 14 mm Botrytis cinerea 33 mm 13 mm

[0302] Table 18 shows that the anti-yeast activity in all fourpreparations is highly heat-resistant. No significant loss of activitywas encountered at heat treatment less than 121° C. TABLE 18 HeatStability of Activity Against Saccharomyces cerevisiae TemperatureCulture Filtrate Fungicide Protease* Comment  30° C. 17 mm 9 mm 16 mm +Control  37° C. 17 mm 9 mm 14 mm +  50° C. 17 mm 9 mm 14 mm +  65° C. 17mm 9 mm 13 mm +  80° C. 16 mm 9 mm 10 mm + 100° C. 16 mm 9 mm 10 mm +121° C.  3 mm 9 mm 10 mm ± Autoclave

[0303] Table 19 shows that Filtrate and Fungicide preparation contain nodetectable antibacterial activity. Further, the activity present in theCulture and Protease preparations is relatively heat sensitive. That is,all activity was lost after incubating at 80° C. for 15 min. TABLE 19Heat Stability of Activity Against Micrococcus luteus TemperatureCulture Filtrate Fungicide Protease* Comment  30° C. 25 mm 0 mm 0 mm +Control  37° C. 25 mm 0 mm 0 mm +  50° C. 19 mm 0 mm 0 mm +  65° C. 18mm 0 mm 0 mm +  80° C.  0 mm 0 mm 0 mm 0 100° C.  0 mm 0 mm 0 mm 0 121°C.  0 mm 0 mm 0 mm 0 Autoclave

[0304] Table 20 shows that the Filtrate preparation contains very littleanti-filamentous fungi activity. Compared to the anti-yeast activity,the anti-filamentous fungi activity is more heat sensitive. Treatment at100° C. for 15 min resulted in a significant loss of activity. TABLE 20Results: Heat Stability of Activity Against Aspergillus nigerTemperature Culture Filtrate Fungicide Protease* Comment  30° C. 28 mm 6mm 13 mm + Control  37° C. 28 mm 0 mm 12 mm +  50° C. 26 mm 0 mm 12 mm + 65° C. 21 mm 0 mm  9 mm +  80° C. 18 mm 0 mm 10 mm + 100° C. 13 mm 0 mm 6 mm + 121° C.  0 mm 0 mm  0 mm 0 Autoclave

[0305] 6.7. Anti-Protozoan Activity of Burkholderia casidae Strain 2.2N

[0306] The experiment described below demonstrates that Burkholderiacasidae strain 2.2N exhibits biocontrol activity against protozoa.

[0307]Tetrahymena pyriformis strain ATCC 30202 was grown in 10 ml ofTetrahymena medium number 357 at 25° C. to early-log phase in 18×150 mmtubes with rotation at 20 rpm. B. casidae strain 2.2 N was grown tomid-log phase in 10 ml TSM+S at 30° C. in a 125 ml Erlenmeyer flask withaeration by rotation at 60 rpm.

[0308] An aliquot of the mid-log B. casidae strain 2.2N culture wasadded to each of four duplicate T pyriformis 10 ml cultures contained in18×150 mm screw capped tubes. Cocultures and B. casidae-free cultures ofT. pyriformis were incubated at 25° C. with 20 rpm.

[0309] Samples of the cultures and cocultures were removed periodicallyfor cell count. The number of strain 2.2N cells in each coculture wasmeasured by plating on TSM+S agar. The plates were incubated at 30° C.The number of strain 2.2N colonies appearing after a few days wascounted in order to determine colony forming unit (CFU) of 2.2N per mlof coculture.

[0310] The number of T. pyriformis cells was determined by visuallycounting the number of cells in dilutions of coculture or culture usinga microscope and a Petroff-Hauser counting chamber. The results arereported as cells per ml of coculture or culture.

[0311] Table 21 shows that coculturing led to an initial decline in thenumber of strain 2.2N cells within the first 12 hr of the coculture. At27 hr after coculturing the number of strain 2.2N cells began to recoverand continued to increase for the next 48 to 72 hours. By contrast, thenumber of T. pyriformis cells in the coculture increased continually forabout 57 hr and then underwent a drastic reduction by 74 hr. No suchreduction was observed for T. pyriformis cultured by itself. Theprecipitous drop in T. pyriformis cell number in the coculture indicatesantagonism and possibly predation by strain 2.2N. TABLE 21 Inhibition ofgrowth of Tetrahymena pyriformis by B. casidae strain 2.2N. T.Pyriformis B. casidae Length of Control Coculture Coculture IncubationCells/ml Cells/ml CFU/ml  0 hr 3.8 × 10⁵ 2.0 × 10⁵ 1.7 × 10⁸ 12 hours6.6 × 10⁵ 5.2 × 10⁵ 9.6 × 10⁶ 27 hours 1.2 × 10⁶ 9.2 × 10⁵ 9.8 × 10⁷ 33hours 1.3 × 10⁶ 1.1 × 10⁶ 6.0 × 10⁷ 50 hours 1.5 × 10⁶ 1.2 × 10⁶ 1.5 ×10⁸ 57 hours 1.5 × 10⁶ 1.2 × 10⁶ 3.9 × 10⁸ 74 hours 1.5 × 10⁶ 3.5 × 10⁴3.3 × 10⁹ 84 hours 1.3 × 10⁶ <2.0 × 10³   3.3 × 10⁹

[0312] Various publications are cited herein, the disclosures of whichare incorporated by reference in their entireties.

[0313] The present invention is not to be limited in scope by thespecific embodiments described herein. Indeed various modifications ofthe invention in addition to those described herein will become apparentto those skilled in the art from the foregoing description andaccompanying figures. Such modifications are intended to fall within thescope of the appended claims.

1 1 1495 base pairs nucleic acid unknown unknown Genomic DNA 1AAATATTACG CTGGTTGCAT GCCTTACAGC ATGCAAGTCG AACGGCAGCA CGGGTGCTT 60CACCTGGTGG CGAGTGGCGA ACGGGTGAGT AATACATCGG AACAATGTCC TGTAGTGG 120GATAGCCCGG CGAAAGCCGG ATTAATACCG CATACGATCT ACGGATGAAA GCGGGGGA 180TTCGGGCCTC GCGCTATAGG GTTGGCCGAT GGCTGATTAG CTAGTTGGTG GGGTAAAG 240CTACCAAGGC GACGATCAGT AGTTGTCTGA GAGGACGACC AGCCACACTG GGACTGAG 300ACGGCCCAGA CTCTTACGGG AGGCAGCAGT GGGGAATTTT GGACAATGGG CGAAAGCC 360ATCCAGCAAT GCCGCGTGTG TGAAGAAGGC CTTCGGGTTG TAAAGCACTT TTGTCCGG 420AGAAATCCTT GGTTCTAATA TAGCCGGGGG ATGACGGTAC CGGAAGAATA AGCACCGG 480AACTACGTGC CAGCAGCCGC GGTAATACGT AGGGTGCGAG CGTTAATCGG AATTACTG 540CGTAAAGCGT GCGCAGGCGG TTTGCTAAGA CCGATGTGAA ATCCCCGGGC TCAACCTG 600AACTGCATTG GTGACTGGCA GGCTAGAGTA TGGCAGAGGG GGGTAGAATT CCACGTGT 660CAGTGAAATG CGTAGAGATG TGGAAGAATA CCGATGGCGA AGGCAGCCCC CTGGGCCA 720ACTGACGCTC ATGCACGAAA GCGTGGGGAG CAAACAGGAT TAGATACCCT GGTAGTCC 780GCCCTAAACG ATGTCAACTA GTTGTTGGGG ATTCATTTCC TTAGTAACGT AGCTAACG 840TGAAGTTGAC CGCCTGGGGA GTACGGTCGC AAGATTAAAA CTCAAAGGAA TTGACGGG 900CCCGCACAAG CGGTGGATGA TGTGGATTAA TTCGATGCAA CGCGAAAAAC CTTACCTA 960CTTGACATGG TCGGAATCCC GCTGAGAGGT GGGAGTGCTC GAAAGAGAAC CGGCGCA 1020GTGCTGCATG GCTGTCGTCA GCTCGTGTCG TGAGATGTTG GGTTAAGTCC CGCAACG 1080GCAACCCTTG TCCTTAGTTG CTACGCAAGA GCACTCTAAG GAGACTGCCG GTGACAA 1140GGAGGAAGGT GGGGATGACG TCAAGTCCTC ATGGCCCTTA TGGGTAGGGC TCACACG 1200TACAATGGTC GGAACAGAGG GTTGCCACCC GCGAAGGGGA GCTAATCCCA GAAAACC 1260CGTAGTCCGG ATTGCACTCT GCACCTCGAG TGCATGAAGC TGGAATCGCT AGTAATC 1320GATCAGCATG CCGCGGTGAA TACTTTCCCG GGTTTTGTAC ACACCGCCCG TCACACC 1380GGAGTGGGTT TTACCAGAAG TGGCTAGTCT AACCGCAAGG AAGAACGGTC CCCACGG 1440GATTCATGAC TGGGTGAAGT CGTAACAAGT AGCCGTATCC GAAAGTTCGG CTGGA 1495

What is claimed is:
 1. A substantially pure culture or suspension of abacterium Burkholderia casidae or variant thereof, which Burkholderiacasidae or variant exhibits biocontrol activity against a microorganism,and has a) a 16S rRNA gene comprising a sequence that is at least 97%similar to the sequence of SEQ ID NO: 1 as determined by ClustalAnalysis; and b) a cellular fatty acid composition comprising about 16%to about 20% C16:0 fatty acid, about 18% to about 22% C16:1 fatty acid,and about 35% to about 45% C18:1 (11,12) fatty acid; wherein themicroorganism is a bacterium, yeast, filamentous fungi, protozoan oralgae.
 2. The substantially pure culture or suspension of claim 1, whichbacterium or variant thereof has a 16S rRNA gene comprising a sequenceidentical to that of SEQ ID NO: 1; and a cellular fatty acid compositioncomprising about 18% C16:0 fatty acid, about 21% C16:1 fatty acid andabout 39% C18:1 (11,12).
 3. A substantially pure culture or suspensionof Burkholderia casidae strain 2.2N having the accession number ATCC55961, or a variant thereof.
 4. The substantially pure culture orsuspension of claim 1, 2 or 3, wherein the culture or suspensioncomprises at least 80% cysts.
 5. The substantially pure culture orsuspension of claim 1, 2 or 3, wherein the culture or suspensioncomprises at least 80% cells.
 6. The substantially pure culture orsuspension of claim 1, 2 or 3, wherein the Burkholderia casidae orvariant thereof has been inactivated.
 7. The substantially pure cultureor suspension of claim 6, wherein the Burkholderia casidae or variantthereof has been inactivated by treating with heat or alcohol.
 8. Acell-free filtrate or cell fraction prepared from the substantially pureculture or suspension of claim 1, 2 or
 3. 9. A cell-free filtrate orcell fraction prepared from the substantially pure culture or suspensionof claim
 6. 10. An antimicrobial preparation comprising analcohol-extract of a cell, cyst, culture, suspension, cell-free filtrateor cell fraction of a bacterium Burkholderia casidae or variant thereof,which Burkholderia casidae or variant exhibits biocontrol activityagainst a microorganism, and has a) a 16S rRNA gene comprising asequence that is at least 97% similar to the sequence of SEQ ID NO: 1 asdetermined by Clustal Analysis; and b) a cellular fatty acid compositioncomprising about 16% to about 20% C16:0 fatty acid, about 18% to about22% C16:1 fatty acid, and about 35% to about 45% C18:1 (11,12) fattyacid; wherein the microorganism is a bacterium, yeast, filamentousfungi, protozoan or algae.
 11. The antimicrobial preparation of claim10, wherein the bacterium or variant thereof has a 16S rRNA genecomprising a sequence identical to that of SEQ ID NO: 1; and a cellularfatty acid composition comprising about 18% C16:0 fatty acid, about 21%C16:1 fatty acid, and about 39% C18:1 (11,12).
 12. The antimicrobialpreparation of claim 11, wherein the bacterium is Burkholderia casidaestrain 2.2N having the accession number ATCC 55961, or a variantthereof.
 13. The antimicrobial preparation of claim 10, 11 or 12,wherein the cell, cyst, culture, suspension, cell-free filtrate or cellfraction of Burkholderia casidae or variant has been inactivated of anylive bacterium.
 14. The antimicrobial preparation of claim 10, 11 or 12,wherein the alcohol-extract is prepared according to a methodcomprising: a) boiling an alcoholic mixture comprising the cell,culture, suspension, cell-free filtrate or cell fraction and an alcohol;b) clarifying the boiled mixture; c) mixing the boiled mixture withmagnesium silicate; d) collecting the magnesium silicate; e) washing themagnesium silicate with water; and f) eluting antifungal compounds fromthe magnesium silicate with an alcoholic solution, thereby producing thealcohol-extract.
 15. A biocontrol composition comprising thesubstantially pure culture or suspension of claim 1, 2 or
 3. 16. Abiocontrol composition comprising the substantially pure culture orsuspension of claim
 4. 17. A biocontrol composition comprising thesubstantially pure culture or suspension of claim
 5. 18. A biocontrolcomposition comprising the substantially pure culture or suspension ofclaim
 6. 19. A biocontrol composition comprising the substantially pureculture or suspension of claim
 7. 20. A biocontrol compositioncomprising sprayed-dried or freeze-dried cells of the substantially pureculture or suspension of claim 1, 2 or
 3. 21. A biocontrol compositioncomprising sprayed-dried or freeze-dried cells of the substantially pureculture or suspension of claim
 4. 22. A biocontrol compositioncomprising sprayed-dried or freeze-dried cells of the substantially pureculture or suspension of claim
 5. 23. A biocontrol compositioncomprising sprayed-dried or freeze-dried cells of the substantially pureculture or suspension of claim
 6. 24. A biocontrol compositioncomprising sprayed-dried or freeze-dried cells of the substantially pureculture or suspension of claim
 7. 25. A biocontrol compositioncomprising the cell-free filtrate or cell fraction of claim
 8. 26. Abiocontrol composition comprising the cell-free filtrate or cellfraction of claim
 9. 27. A biocontrol composition comprising theantimicrobial preparation of claim
 10. 28. A biocontrol compositioncomprising the antimicrobial preparation of claim
 11. 29. A biocontrolcomposition comprising the antimicrobial preparation of claim
 12. 30. Abiocontrol composition comprising the antimicrobial preparation of claim13.
 31. A biocontrol composition comprising the antimicrobialpreparation of claim
 14. 32. A method for treating or preventing adisease of a plant, comprising applying an effective amount of thebiocontrol composition of claim 15 to the plant.
 33. A method fortreating or preventing a disease of a plant, comprising applying aneffective amount of the biocontrol composition of claim 18 to the plant.34. A method for treating or preventing a disease of a plant, comprisingapplying an effective amount of the biocontrol composition of claim 20to the plant.
 35. A method for treating or preventing a disease of aplant, comprising applying an effective amount of the biocontrolcomposition of claim 23 to the plant.
 36. A method for treating orpreventing a disease of a plant, comprising applying an effective amountof the biocontrol composition of claim 25 to the plant.
 37. A method fortreating or preventing a disease of a plant, comprising applying aneffective amount of the biocontrol composition of claim 26 to the plant.38. A method for treating or preventing a disease of a plant, comprisingapplying an effective amount of the biocontrol composition of claim 27to the plant.
 39. A method for treating or preventing a disease of aplant, comprising applying an effective amount of the biocontrolcomposition of claim 28 to the plant.
 40. A method for treating orpreventing a disease of a plant, comprising applying an effective amountof the biocontrol composition of claim 29 to the plant.
 41. A method fortreating or preventing a disease of a plant, comprising applying aneffective amount of the biocontrol composition of claim 30 to the plant.42. A method for treating or preventing a disease of a plant, comprisingapplying an effective amount of the biocontrol composition of claim 31to the plant.
 43. The method according to claim 32, wherein themicroorganism is a species of a genus selected from the group consistingof Alternaria, Aspergillus, Botrytis, Cercospora, Cercosporidium,Geotrichum, Mycosphaerella, Mucor, Penicillium, Phoma, Phytophthora,Plasmopora, Pseudopeziza, Puccinia, Pythium, Rhizoctonia, Rhizopus,Saccharomyces, Septoria, Sporothrix, Stemphylium, Trichophyton,Verticillium, Erwinia, Pseudomonas and Xanthomonas.
 44. The methodaccording to claim 33, wherein the microorganism is a species of a genusselected from the group consisting of Alternaria, Aspergillus, Botrytis,Cercospora, Cercosporidium, Geotrichum, Mycosphaerella, Mucor,Penicillium, Phoma, Phytophthora, Plasmopora, Pseudopeziza, Puccinia,Pythium, Rhizoctonia, Rhizopus, Saccharomyces, Septoria, Sporothrix,Stemphylium, Trichophyton, Verticillium, Erwinia, Pseudomonas andXanthomonas.
 45. The method according to claim 34, wherein themicroorganism is a species of a genus selected from the group consistingof Alternaria, Aspergillus, Botrytis, Cercospora, Cercosporidium,Geotrichum, Mycosphaerella, Mucor, Penicillium, Phoma, Phytophthora,Plasmopora, Pseudopeziza, Puccinia, Pythium, Rhizoctonia, Rhizopus,Saccharomyces, Septoria, Sporothrix, Stemphylium, Trichophyton,Verticillium, Erwinia, Pseudomonas and Xanthomonas.
 46. The methodaccording to claim 35, wherein the microorganism is a species of a genusselected from the group consisting of Alternaria, Aspergillus, Botrytis,Cercospora, Cercosporidium, Geotrichum, Mycosphaerella, Mucor,Penicillium, Phoma, Phytophthora, Plasmopora, Pseudopeziza, Puccinia,Pythium, Rhizoctonia, Rhizopus, Saccharomyces, Septoria, Sporothrix,Stemphylium, Trichophyton, Verticillium, Erwinia, Pseudomonas andXanthomonas.
 47. The method according to claim 36, wherein themicroorganism is a species of a genus selected from the group consistingof Alternaria, Aspergillus, Botrytis, Cercospora, Cercosporidium,Geotrichum, Mycosphaerella, Mucor, Penicillium, Phoma, Phytophthora,Plasmopora, Pseudopeziza, Puccinia, Pythium, Rhizoctonia, Rhizopus,Saccharomyces, Septoria, Sporothrix, Stemphylium, Trichophyton,Verticillium, Erwinia, Pseudomonas and Xanthomonas.
 48. The methodaccording to claim 37, wherein the microorganism is a species of a genusselected from the group consisting of Alternaria, Aspergillus, Botrytis,Cercospora, Cercosporidium, Geotrichum, Mycosphaerella, Mucor,Penicillium, Phoma, Phytophthora, Plasmopora, Pseudopeziza, Puccinia,Pythium, Rhizoctonia, Rhizopus, Saccharomyces, Septoria, Sporothrix,Stemphylium, Trichophyton, Verticillium, Erwinia, Pseudomonas andXanthomonas.
 49. The method according to claim 38, wherein themicroorganism is a species of a genus selected from the group consistingof Alternaria, Aspergillus, Botrytis, Cercospora, Cercosporidium,Geotrichum, Mycosphaerella, Mucor, Penicillium, Phoma, Phytophthora,Plasmopora, Pseudopeziza, Puccinia, Pythium, Rhizoctonia, Rhizopus,Saccharomyces, Septoria, Sporothrix, Stemphylium, Trichophyton,Verticillium, Erwinia, Pseudomonas and Xanthomonas.
 50. The methodaccording to claim 39, wherein the microorganism is a species of a genusselected from the group consisting of Alternaria, Aspergillus, Botrytis,Cercospora, Cercosporidium, Geotrichum, Mycosphaerella, Mucor,Penicillium, Phoma, Phytophthora, Plasmopora, Pseudopeziza, Puccinia,Pythium, Rhizoctonia, Rhizopus, Saccharomyces, Septoria, Sporothrix,Stemphylium, Trichophyton, Verticillium, Erwinia, Pseudomonas andXanthomonas.
 51. The method according to claim 40, wherein themicroorganism is a species of a genus selected from the group consistingof Alternaria, Aspergillus, Botrytis, Cercospora, Cercosporidium,Geotrichum, Mycosphaerella, Mucor, Penicillium, Phoma, Phytophthora,Plasmopora, Pseudopeziza, Puccinia, Pythium, Rhizoctonia, Rhizopus,Saccharomyces, Septoria, Sporothrix, Stemphylium, Trichophyton,Verticillium, Erwinia, Pseudomonas and Xanthomonas.
 52. The methodaccording to claim 41, wherein the microorganism is a species of a genusselected from the group consisting of Alternaria, Aspergillus, Botrytis,Cercospora, Cercosporidium, Geotrichum, Mycosphaerella, Mucor,Penicillium, Phoma, Phytophthora, Plasmopora, Pseudopeziza, Puccinia,Pythium, Rhizoctonia, Rhizopus, Saccharomyces, Septoria, Sporothrix,Stemphylium, Trichophyton, Verticillium, Erwinia, Pseudomonas andXanthomonas.
 53. The method according to claim 42, wherein themicroorganism is a species of a genus selected from the group consistingof Alternaria, Aspergillus, Botrytis, Cercospora, Cercosporidium,Geotrichum, Mycosphaerella, Mucor, Penicillium, Phoma, Phytophthora,Plasmopora, Pseudopeziza, Puccinia, Pythium, Rhizoctonia, Rhizopus,Saccharomyces, Septoria, Sporothrix, Stemphylium, Trichophyton,Verticillium, Erwinia, Pseudomonas and Xanthomonas.
 54. The methodaccording to claim 32, wherein the plant is selected from the groupconsisting of safflower, cotton, flax, oats, canola, poinsettia,chrysanthemum, corn, soybean, wheat, rice, alfalfa, sorghum, peanut,tobacco, tomato, pepper, cucumber, lettuce, green bean, lima beans,peas, cantaloupe, musk melon, citrus fruit, grape, banana, geranium,azalea, rose, tulip, petunia, orchid, carnation, pine, yew and spruce.55. The method according to claim 33, wherein the plant is selected fromthe group consisting of safflower, cotton, flax, oats, canola,poinsettia, chrysanthemum, corn, soybean, wheat, rice, alfalfa, sorghum,peanut, tobacco, tomato, pepper, cucumber, lettuce, green bean, limabeans, peas, cantaloupe, musk melon, citrus fruit, grape, banana,geranium, azalea, rose, tulip, petunia, orchid, carnation, pine, yew andspruce.
 56. The method according to claim 34, wherein the plant isselected from the group consisting of safflower, cotton, flax, oats,canola, poinsettia, chrysanthemum, corn, soybean, wheat, rice, alfalfa,sorghum, peanut, tobacco, tomato, pepper, cucumber, lettuce, green bean,lima beans, peas, cantaloupe, musk melon, citrus fruit, grape, banana,geranium, azalea, rose, tulip, petunia, orchid, carnation, pine, yew andspruce.
 57. The method according to claim 35, wherein the plant isselected from the group consisting of safflower, cotton, flax, oats,canola, poinsettia, chrysanthemum, corn, soybean, wheat, rice, alfalfa,sorghum, peanut, tobacco, tomato, pepper, cucumber, lettuce, green bean,lima beans, peas, cantaloupe, musk melon, citrus fruit, grape, banana,geranium, azalea, rose, tulip, petunia, orchid, carnation, pine, yew andspruce.
 58. The method according to claim 36, wherein the plant isselected from the group consisting of safflower, cotton, flax, oats,canola, poinsettia, chrysanthemum, corn, soybean, wheat, rice, alfalfa,sorghum, peanut, tobacco, tomato, pepper, cucumber, lettuce, green bean,lima beans, peas, cantaloupe, musk melon, citrus fruit, grape, banana,geranium, azalea, rose, tulip, petunia, orchid, carnation, pine, yew andspruce.
 59. The method according to claim 37, wherein the plant isselected from the group consisting of safflower, cotton, flax, oats,canola, poinsettia, chrysanthemum, corn, soybean, wheat, rice, alfalfa,sorghum, peanut, tobacco, tomato, pepper, cucumber, lettuce, green bean,lima beans, peas, cantaloupe, musk melon, citrus fruit, grape, banana,geranium, azalea, rose, tulip, petunia, orchid, carnation, pine, yew andspruce.
 60. The method according to claim 38, wherein the plant isselected from the group consisting of safflower, cotton, flax, oats,canola, poinsettia, chrysanthemum, corn, soybean, wheat, rice, alfalfa,sorghum, peanut, tobacco, tomato, pepper, cucumber, lettuce, green bean,lima beans, peas, cantaloupe, musk melon, citrus fruit, grape, banana,geranium, azalea, rose, tulip, petunia, orchid, carnation, pine, yew andspruce.
 61. The method according to claim 39, wherein the plant isselected from the group consisting of safflower, cotton, flax, oats,canola, poinsettia, chrysanthemum, corn, soybean, wheat, rice, alfalfa,sorghum, peanut, tobacco, tomato, pepper, cucumber, lettuce, green bean,lima beans, peas, cantaloupe, musk melon, citrus fruit, grape, banana,geranium, azalea, rose, tulip, petunia, orchid, carnation, pine, yew andspruce.
 62. The method according to claim 40, wherein the plant isselected from the group consisting of safflower, cotton, flax, oats,canola, poinsettia, chrysanthemum, corn, soybean, wheat, rice, alfalfa,sorghum, peanut, tobacco, tomato, pepper, cucumber, lettuce, green bean,lima beans, peas, cantaloupe, musk melon, citrus fruit, grape, banana,geranium, azalea, rose, tulip, petunia, orchid, carnation, pine, yew andspruce.
 63. The method according to claim 41, wherein the plant isselected from the group consisting of safflower, cotton, flax, oats,canola, poinsettia, chrysanthemum, corn, soybean, wheat, rice, alfalfa,sorghum, peanut, tobacco, tomato, pepper, cucumber, lettuce, green bean,lima beans, peas, cantaloupe, musk melon, citrus fruit, grape, banana,egeranium, azalea, rose, tulip, petunia, orchid, carnation, pine, yewand spruce.
 64. The method according to claim 42, wherein the plant isselected from the group consisting of safflower, cotton, flax, oats,canola, poinsettia, chrysanthemum, corn, soybean, wheat, rice, alfalfa,sorghum, peanut, tobacco, tomato, pepper, cucumber, lettuce, green bean,lima beans, peas, cantaloupe, musk melon, citrus fruit, grape, banana,geranium, azalea, rose, tulip, petunia, orchid, carnation, pine, yew andspruce.
 65. A method for producing an antimicrobial preparation fromBurkholderia casidae or variant thereof, comprising: a) boiling analcoholic mixture comprising a cell, culture, suspension, cell-freefiltrate or cell fraction of Burkholderia casidae or variant thereof,and an alcohol; b) clarifying the boiled mixture; c) mixing the boiledmixture with magnesium silicate; d) collecting the magnesium silicate;e) washing the magnesium silicate with water; and f) eluting antifungalcompounds from the magnesium silicate with an alcoholic solution,thereby producing the antimicrobial preparation.
 66. A method forisolating a Burkholderia casidae or variant thereof, which comprises (a)determining the 16S rRNA sequence and the cellular fatty acidcomposition of a predator bacterium which exhibits biocontrol activityagainst a microorganism; and (b) identifying a bacterium having (i) a16S rRNA gene comprising a sequence that is at least 97% similar to thesequence of SEQ ID NO: 1 as determined by Clustal Analysis, and (ii) acellular fatty acid composition comprising about 16% to about 20% C16:0fatty acid, about 18% to about 22% C16:1 fatty acid, and about 35% toabout 45%C 18:1 (11,12) fatty acid; thereby isolating the Burkholderiacasidae or variant thereof.
 67. The method of claim 66, wherein thebacterium has (i) a 16S rRNA gene comprising a sequence that isidentical to the sequence of SEQ ID NO: 1, and (ii) a cellular fattyacid composition comprising about 18% C16:0 fatty acid, about 21% C16:1fatty acid and about 39% C18:1 (11,12).